Rhizobium under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few Rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. MK and UQ were extracted from the wild type strain 7653R and mutant strain HK116. MK-7 was accumulated under microaerobic condition and UQ-10 was accumulated under aerobic condition in M. huakuii 7653R. The predicted function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results revealed that MK-7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids.Ubiquinone (UQ) has been considered as an electron mediator in electron transfer that generates ATP in  E. coli and closely related species can switch between MK-and UQ-based bioenergetic chains in response to varying growth conditions. In E. coli, UQ and MK perform distinct functions. UQ-8 is used as the main quinone in aerobic respiration In bioenergetic chains, diverse chemical types of quinones, such as ubi-, plasto-, mena-, rhodo-, caldariella- or sulfolobus-quinones have been identified as membrane-bound, mobile hydrogen carriers in different species. Ubiquinone (UQ) is the dominant quinone species in α/β/γ-proteobacteria. The majority of strictly aerobic Gram-negative bacteria contain exclusively UQ menG genes . SHCHC is dehydrated into o-uccinylbenzoate, and linked with CoA, then converted into 1,4-dihydroxy-2-naphthoate(DHNA). DHNA is added prenyl side chain into DMK, and DMK is converted into MK by S-adenosylmethionine-dependent methylationnG genes . RecentlRhizobium, classfied as the Gram-negative α-proteobacteria, is strictly aerobic under free-living but is microaerobic as bacteroids in nodules. Rhizobium contains diverse and complicated respiratory electron transport systems to cope with free-living and symbiosis conditions. Under aerobic conditions, BradyRhizobium japonicum transfers electrons from the ubiquinone (UQ) pool to the O2-reducing aa3-type terminal oxidase via the FeS protein-cytochrome bc1 complex and a membrane-bound cytochrome cB. japonicum, and one of the mainstream chains was that a cbb3-type-terminal oxidase transferred electrons from Fes/bc1 to O2bc1Rhizobium trifolii, flavoprotein lies between NADH and UQ, and b-cytochromes transfer electrons from UQ to O2Rhizobium cultured under different conditions.Mesorhizobium huakuii can establish a nitrogen-fixing symbiosis with Astragalus sinicus and form indeterminate nodules. M. huakuii has a strict specificity for A. sinicus which makes this species important for studies on the molecular mechanism of symbiotic nitrogen fixation. We previously constructed a transposon mutagenesis library by random insertion of the Tn5-B12S into the M. huakuii 7653R genome dmtH, was isolated from the mutagenesis library which mutant formed ineffective nodules on A. sinicus. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes DMK methyltransferase, which catalyzes the terminal step of MK biosynthesis. The result is interesting,because no Rhizobium has been reported to operate electron transfer containing both MK and UQ thus far. We hypothesized that a few of Rhizobia may use MK as electron carrier under symbiosis conditions. In this study, we identified dmtH function by symbiotic phenotype and protein enzymatic activity assays in vitro. The results suggested that dmtH encoded a DMK methyltransferases and MK was biosynthesized in M. huakuii bacteroids. The implications of these findings for the functions of dmtH and MK in bacteroid metabolism and symbiosis are also discussed.+fix−). After sequencing, a homology search revealed that a consensus sequence was present only in some isolates of Rhizobium. Since the genome of M. huakuii has not still been sequenced, a pair of specific primers, dmtUP and dmtLOW were designed, which were based on the homologous sequences of M. loti MAFF303099, to amplify a 750-bp fragment including the complete ORF sequences from the genome of wild-type strain 7653R. This gene was designated as dmtH (GenBank accession no. JN400272). The dmtH gene is predicted to encode a 249-amino acid polypeptide with an expected molecular mass of 27.3 kDa and a pI value of 5.4. The homology analysis of the DmtH protein showed that had 99% identity to the function-unknown gene mll2332 from M. loti MAFF303099, and another similar proteins were annotated as demethylmenaquinone methyltransferase proteins in M. opportunistum WSM2075 (94% identity), R. leguminosarum bv. trifolii WSM1325 (90% identity) and R. etli CFN42 (88% identity). DmtH homology protein also presented in other bacteria as annotated demethylmenaquinone methyltransferase, such as Amycolatopsis mediterranei U32 (59% identity), Roseiflexus sp. RS-1 (49% identity) . We thus assumed that the DmtH protein encoded DMK methyltransferase, which catalyzes methylation at the terminal step in MK biosynthesis.The flanking sequence of the transposon insertion site was amplified by TAIL-PCR, from the mutant strain HK115 (noddentity) , S2. ThedmtH gene in nodulation and nitrogen fixation ability was disrupted by the transposon insertion, a dmtH gene null mutant was constructed by the homologous recombination method, and one mutant strain, HK116, which formed ineffective nodules on A. sinicus, was obtained. The disruption of dmtH in mutant strain HK116 was verified by PCR. The symbiotic phenotype of the disruptant HK116 was examined by plant growth test . These results indicated that dmtH gene expression was induced by symbiosis, and was indispensable for bacteroid development and nitrogen fixation, but was not needed for the free-living growth and metabolism of Rhizobia.The relative expression level of the time PCR . The resRhizobium. The function of DmtH protein was hypothesized to participate in the biosynthesis of MK in 7653R, and the dmtH gene was specifically expressed in bacteroids. Therefore, we compared and identified the electron carrier used under free-living and symbiotic conditions. Quinone was extracted from 7653R and mutant HK116 growing under aerobic and microaerobic conditions and extracts were analyzed by reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The results demonstrated that UQ-10 accumulated in 7653R and HK116 under aerobic conditions, but when growing conditions were switched from aerobic to microaerobic, MK-7 was confirmed to be produced in the wild type strain 7653R but was not in the extract from mutant HK116, in which the content of UQ-10 was also significantly decreased . The target protein was overexpressed after inducing with isopropyl β-D-thiogalactopyranoside and purified  was used as the methyl donor. The reaction system, with the crude E.coli JC7623Δ4-1 homogenate, purified DmtH protein and SAM, was incubated at 37°C for 1 h, and the products analyzed by HPLC and MS. MK-8 was found in the reaction products in addition to DMK-8  medium at 37°C. Rhizobia were grown in tryptone yeast medium (TY) at 28°C. When required, the following antibiotics were used: ampicillin (Ap) 100 µg/ml; spectinomycin (Spe) 50 µg/ml; tetracycline (Tet) 15 µg/ml; gentamicin (Gm) 20 µg/ml; and kanamycin (Kan) 50 µg/ml.M. huakuii has not still been sequenced at present, hence thermal asymmetric interlaced PCR (TAIL-PCR) was used to amplify the flanking regions of Tn5-B12S from the genome of the original symbiosis defective mutant HK115 (nod+fix−). Techniques were performed as previously described dmtH (GenBank accession no. JN400272). Two oligonucleotides, dmtUP (5′-ATGACGCATACCTCCGACATC-3′) and dmtLOW (5′-CTAATTTGGGTTCCCCAAGC-3′) were designed to amplify the 750-bp DNA sequences containing the full-length ORF of dmtH gene from total DNA of M. huakuii 7653R. Nucleotides and protein homology searches were carried out by BLAST analysis (http://www. ncbi. nlm. nih.gov/BLAST). The protein-conserved domains were predicted using the InterProScan server of EBI (http://www.ebi.ac.uk/InterProScan).The genome of dmtH ORF with 200 bp of flanking sequence on either side of the coding sequence were: dmtUP1: 5′-TCAATTTCAACCGGAGTCC-3′; dmtLOW1: 5′-GACCTGGTTTCGCGCGCG-3′. The resulting PCR product (1.1 kb) was ligated into pMD-18T to generate pMDdmtH. A 950-bp fragment containing a Km-resistance cassette was amplified from plasmid pMH1701 by PCR with primers kanNcoIU  and kanNcoIL . The Km-resistance cassette was inserted into the unique NcoI site of the dmtH gene in pMDdmtH to create plasmid pMDdmtH-Km. A 2.1-kb fragment containing dmtH and the Km cassette was cut from pMDdmtH-Km and subcloned into pJQ200 mp18 using PstI and XbaI restriction sites. The resulting suicide plasmid, pJQdmtH-Km, was conjugated into M. huakuii 7653R by tri-parental mating with a helper plasmid pRK2013 dmtH double-crossover recombinant was selected using the sac mutagenesis strategy as previously described dmtH in mutant strain HK116 was verified by PCR and plant nodulation experiment.Primers used to PCR-amplify a segment of 7653R genomic DNA containing the dmtH gene was amplified with 7653R total DNA as the template using the following two primers: dmtUP2: CTCGAGTAATCTAGACTGAACGAGGAGGTCGACGATGACGCATAC-3′5′- ACT (the XhoI restriction site is underlined), dmtLOW2: CTCGAGCTAATTTGGGTTCCCCAA -3′5′- ACT (the XhoI restriction site is underlined). The amplified fragment digested with XhoI was cloned into the previously modified vector pBBRgus, in which gusA was inserted into the broad-host-range plasmid pBBR1MCS5 under the Lac promoter gus-dmt was obtained, and transferred into mutant strain HK116 by triparental mating. Transconjugants were used to inoculate seedlings of A. sinicus to test for ability to restore the symbiotic phenotype of HK116.For the symbiotic function complementation test, the A. sinicus cultivar XY202 were surface sterilized for 5 min in 75% ethanol, soaked for 20 min in 2% sodium hypochlorite, and rinsed extensively with sterile water. Plants were grown in pots filled with sterile sand containing nitrogen-free Fahraeus solution Seeds of Light and transmission electron microscopic studies of nodules were performed as follows. For paraffin sections, root nodules were dehydrated in ethanol and xylene, embedded in paraffin wax, serial-cut longitudinally, and slides were stained with toluidine blue, then observed with a light microscope. For electron microscopy, after nodules were fixed with 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, rinsed, dehydrated and embedded in London resin white, ultra-thin sections were taken and examined in a Hitachi H-7650 transmission electron microscope.dmtH gene expression level, total RNA was isolated with Trizol reagent from free-living M. huakuii 7653R cultured in TY liquid medium and plant nodules, which were harvested from A. sinicus inoculated with wild type strain 7635R after 10, 15, 20, 25, 30, 35 and 40 days. Quantitative real-time PCR was performed using the SYBR Premix ExTaq  on the iCycler iQ5 Multicolor Real-Time PCR Detection System (primers: 5′-CATCTATCCCAATGCCGTCAAC-3′ and 5′-CGAGAACTCTTCCCAATCGTG-3′) . Expression levels were normalized using rnpB gene as internal control (primers: 5′- AAGGCCGCAAGTGAGGAAAGTC-3′ and 5′-GGTTTACCGTGCCGCTCCTGTTG-3′). Data were analyzed by the relative quantification method (2−ΔΔCT) to calculate expression and reactions were performed in triplicate.To determine the relative 2 by sealing the flask with a stopper through which a cannula was inserted to allow continuous sparging of the culture with a compressed gas mixture of prepurified N2 (>99%), the indicated amount of O2, and 10 mM KNO3, which was added to the culture under microaerobic conditions. After culturing for 36 hours microaerobically, cells were harvested by centrifugation, and quinones were extracted with methanol/ethyl acetate (1∶1) and evaporated in vacuo Cells grown for extraction were cultured aerobically in a 500-ml flask containing 100 ml of basal medium  at 28°C and 200 rpm for 20 h, then shifted to low Odmt for the dmtH gene. Two oligonucleotide primers TCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACGATGACGCATACC-3′5′-CTCGAG (the XbaI restriction site is underlined) and CTCGAGATTTGGGTTCCCCAA-3′5′- ATAACT (the XhoI restriction site is underlined) were synthesized, and used to amplify dmtH. PCR products were cloned into the pET28a vector digested with XbaI and XhoI. The resulting plasmid, named pETdmt, was transformed into E. coli Rosetta 2(DE3) for the overexpression of the target protein DmtH. Expression, extraction and purification of DmtH protein was carried out by standard methods on Bio-Rad Biologic LP apparatus To obtain adequate amounts of target protein for activity measurements, we constructed the overexpression plasmid pETE. coli JC7623Δ4-1, which contains only DMK but no MK, was kindly supplied by Professor Catherine F. Clarke of University of California. Crude homogenates of E. coli JC7623Δ4-1 cells were prepared as a reaction substrate for DmtH protein activity assays. As described previously, JC7623Δ4-1 cells grown into late exponential phase in LB medium were centrifuged, suspended in a solution of 25 mM Tris-HCl buffer pH 7.7 containing 1 mM EDTA and 10 mM 2-mercaptoethanol (2 ml/g of wet cells), and disrupted with a Branson sonifier Δ4-1 cells, 0.1 M Tris-HCl buffer (pH 8.0), 10 mM MgCl2, 10 mM dithiothreitol, 10% aqueous Triton X-100 , 10 nmol S-adenosyl-L-methionine (SAM) and 1 µM dmtH product. After incubation at 37°C for 1 h, the reaction was stopped by addition of 10 ml of 0.1 M acetic acid in methanol. The reaction products were extracted with 40 ml methanol/chloroform (1∶1) and evaporated in vacuo. The lipid extracts were resuspended in 0.5 ml of chloroform/liter of culture, and analyzed by HPLC and MS.The DmtH protein reaction mixture contained, in a total volume of 10 ml, 4 ml of crude homogenate of JC7623Figure S1Multiple sequence alignment of the DmtH protein and the homologous sequence. Columns that are dark represent identical residues, lighter black represent similar residues in function. The numbers on the upper indicate the positions of amino acids. Protein names are indicated to the left of the alignment. These proteins come from Moserhizobium. loti MAFF303099 (mlr2332), Moserhizobium opportunistum WSM2075 (MesopDRAFT_4419), Rhizobium leguminosarum bv. viciae WSM1325 (RLeq4868), Rhizobium etli CFN42 (RHE_PE00095), Amycolatopsis mediterranei U32 (AMED3148), Roseiflexus sp. RS-1 (RoseRS_2046), Thermomicrobium roseum DSM 5159 (trd_1944), Roseiflexus castenholzii DSM 13941 (Rcas_3023), Streptomyces lividans TK24 (SlivT_010100003505), Streptomyces ghanaensis ATCC 14672 (SSFG_00363), Burkholderia phytofirmans PsJN (Bphyt_4892).(TIF)Click here for additional data file.Figure S2Homology tree of DmtH homologs. The homology tree was constructed using MEGA5.04 software . The scale bar indicates 10% substitutions per site.(TIF)Click here for additional data file.Figure S3Southern blot analysis of dmtH gene in different Rhizobium genomes. The total genomic DNA of tested strains were digested for at least 3 h with 5-10 U of restriction enzyme PstI and BamHI per µg of DNA. Digested genomes were subjected to electrophoresis and transfer to nylon membranes, then hybridized to a 32P-labeled probe. The tested strains was Agrobacterium tumefaciense IAMI 3129 (A. tum), Sinorhizobium medicae USDA 1037 (S. med), Sinorhizobium meliloti USDA 2011 (S. mel), Sinorhizobium saheli LMG 7837 (S. sah), Rhizobium leguminosarum LRP 5045 (R. leg), Sinorhizobium fredii HN01 (S. fre), Mesorhizobium loti 541 (M. lot), Sinorhizobium arboris HAMBI 1552(S. arb), Azorhizobium caulinodans USDA 4892 (A. cau). (A) the electrophoresis map of BamHI-digested genomic DNA. (B) the electrophoresis map of PstI-digested genomic DNA. (C) BamHI-digested DNA hybridised with probe dmtH. (D) PstI-digested DNA hybridised with probe dmtH. The Southern blot shows that dmtH gene is present in R. leguminosarum LRP 5045 and M. loti 541.(TIF)Click here for additional data file.Figure S4HPLC of the pure standard MK-4, MK-7, MK-9 and UQ-10.(TIF)Click here for additional data file.Figure S5Mass spectrum of quinine compounds from E.coli JC7623Δ4-1 and the reacting system of testing DmtH peotein activity. (A) Mass spectrum of DMK-8 which was accumulated in E.coli JC7623Δ4-1 but no MK-8′ MS found. (B) Mass spectrum of MK-8 which was tested from in reaction system. The result showed MK-8 was synthesized in the reaction system and DmtH had the the function of methyltransferase.(TIF)Click here for additional data file.


Transcription initiation RNAs (tiRNAs) are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII) transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation.p21 . Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21  transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis.In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF) binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization. In addition to mRNAs, it is now clear that most eukaryotic genic loci generate a complex network of overlapping short (<200) and long non-protein coding RNA (ncRNA) species -3. This CTCF is a highly conserved zinc finger protein associated with a diverse set of phenomena including epigenetic insulation, imprinting and transcriptional regulation -13. Intrp21  [p16 (INK4a), the latter by insulating the promoter from silent-state histone modifications such as H3K27 trimethylation (H3K27me3) [frataxin (FXN), a gene mutated and silenced in Friedreich ataxia, which causes progressive damage to the nervous system [FXN leads to a deficiency of the FXN transcript, an increase in FXN antisense transcript 1, and heterochromatin formation involving the +1 nucleosome [CTCF has been shown to regulate the expression of several tumor suppressor genes, including  CDKN1A) ,15 and ps system . Loss ofcleosome . Given tcleosome -22, we sWe have previously shown that tiRNAs isolated from THP-1 cells (a human monocytic leukemia cell line) are systematically enriched at white blood cell CTCF binding sites . To examConsistent with prior work we found that tiRNAs derived from both MCF-7 and mESCs are enriched approximately sixfold at CTCF binding sites that sit outside TSSs or other annotated genomic features  ,24 and Rp21, which is conserved across both multiple human cell types  that originate from a unique promoter located approximately 2 kb upstream of the canonical p21 transcription start site and include the majority of the p21 coding regions in their final spliced products [bx332409) that regulates local epigenetic states [To experimentally interrogate the tiRNA-CTCF-RNAPII relationship we queried for sites in clinically relevant genes and identified a CTCF-RNAPII site with tiRNAs in the first intron of products , and a lc states   and the other at the CTCF-RNAPII site -tiRNAs) that are antisense to one another. The tss-tiRNAs are sense to the gene , while the cp-tiRNAs are antisense. Both overlap distinct peaks of RNAPII binding, suggesting that their biogenesis is tied to RNAPII molecules heading in opposite directions, possibly linked to nucleosome position [The position , and thip21 mRNA and alt p21expression, as measured by quantitative PCR (qPCR)  Figure . In contn Figure , and thup21 transcript levels, strand-specific reverse transcription PCR (RT-PCR) was performed. Upon treatment with the cp-tiRNA sponge, p21 mRNA, sense altp21, and antisense alt p21transcript levels increased, whereas transcripts antisense to p21 mRNA were unaffected . The only signal increase appeared in regions overlapping t Figure , suggestp21 locus by ChIP. The density of the silent state chromatin mark, H3K27me3, did not change upon introduction of cp-tiRNA sponge or mimic constructs at their perfectly complementary target sites ordering of the p21 locus. Indeed, western blot analysis showed that p21 protein levels were increased in samples treated with the cp-tiRNA sponge, and decreased in samples treated with the cp-tiRNA mimic constructs , and an intergenic site upstream of StAR-related lipid transfer domain containing 13 (STARD13)  or polycomb group components and direct their action to specific sites; or (iv) cp-tiRNAs may serve as sequence-specific markers for chromatin modification complexes.The mechanism by which tiRNAs inhibit CTCF localization is unclear, although there are several obvious possibilities: (i) cp-tiRNAs spanning the CTCF binding site may coat local chromatin by binding nascent transcripts  or chromp21 not only reduces p21 mRNA and protein levels, but also appears to affect chromatin state and expression of alt p21transcripts. This suggests that at some loci the role of tiRNAs, whose biogenesis is connected to RNAPII activity and progression, may be to modulate (presumably indirectly) local chromatin states, which in turn regulates the binding of other factors including CTCF. Indeed, the relationship between tiRNAs and epigenetic structures may indicate a self-reinforcing feedback loop wherein the RNAPII-nucleosome interaction generates tiRNAs, which in turn serve to mark (directly or indirectly) nucleosome positions and/or epigenetic state. Although the mechanism of tiRNA action is still elusive, this work is the first to report a role for tiRNAs in gene regulation, and shows that at least a subset of tiRNAs are functional modulators of CTCF, which may lead to the development of novel RNA-based therapeutics that target epigenetic regulation of gene structure and transcription.The data presented here indicate that cp-tiRNAs can have a powerful effect on CTCF binding and local transcription. Indeed, tiRNA-mediated modulation of CTCF binding at Bioinformatic analyses were performed on a local high-performance computer at the UQ Institute for Molecular Bioscience that houses a mirror of the UCSC Genome Brower . We used2 in Dulbecco's modified Eagle's medium or RPMI 1640 (THP-1 cells only) supplemented with 10% fetal bovine serum and penicillin-streptomycin . Plasmid transfections of MCF-7 cells were performed using Lipofectamine 2000  per the manufacturer's guidelines at a concentration of 1 μg/1 × 106 cells. Plasmid transfections of THP-1 cells were carried out using the Neon electroporation system , per manufacturer's guidelines.Experiments were conducted in MCF-7 and THP-1 cells cultured at 37°C and 5% COBglII restriction sites on either end  5'CCGCCGGCCCGGGGTCCCCTGTTGTCTGCCGCCGCTCTCTCACCTCCTCTGAGTGCCTCGGT GCCTCGGCGAATCCGCGCCCAGCTCCGGCTCCACAAGGAACTGACTTCGGCAGCTGCTCACA CCTCAGCTGG3' and p21 CTCF-proximal tiRNA sponge (cp-tiRNA) 5'GGGCTCAGGGCTTCCCGACCTAGCCCAGATTCCCTCTCCGAAAGCTACAGGGCTGAGCGGA GCAGGGGGGCGAGTCGCCCCCTGGGGCGCCGCCGCCTGGCGCGGACCACAGCGCGTCCTCT CCGTCCCAAACCCCTGGGGGACACTTGCGCCCTCTTCGTGAGGAAAAGCATCTTGGAGCTGG GTTAGGAACTTGGGGCGCCCAGGCAGCTTCCCCTCTCCTTGCCTCCCTCCACGTCGCGTTTCT GGGAGGACTTGCGAGCGGTTTTGTTTTCGTTGCTCCCGTCTATTTTTATTTTCCAGGGATCTGA CT3'.Sponge plasmids: reverse complement sequences to the promoter-proximal and CTCF-proximal tiRNA sites in p21 were amplified from MCF-7 genomic DNA by polymerase chain reaction (PCR) using primers with Sponge constructs for C2Orf41 and STARD13 were constructed by annealing the respective oligonucleotides together  and cloned into the BLOCK-iT U6 RNAi Entry Vector  per manufacturer's guidelines. The mimics, four predominant cp-tiRNA sequences , DNase treated , reverse transcribed  using the non-specific or indicated primers (for strand-specific RT-PCR), and analyzed by qPCR using indicated primers   and separated on a NuPAGE 4% to 12% BisTris gel . Proteins were transferred to a nitrocellulose membrane which was blocked with 5% milk for 1 h and then incubated overnight at 4°C with anti-p21  and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  antibodies. The membrane was then washed  and incubated with an anti-mouse horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature . The membrane was then washed, treated with chemiluminescent detection reagent , and exposed to film. Blot density of a binary image of Figure RJT and JSM have an ownership stake in a patent concerning tiRNAs and their diagnostic and therapeutic uses .RJT performed the bioinformatic analyses, helped design the laboratory experiments and wrote the manuscript. PGH performed the generated and tested tiRNA mimics and sponges and helped write the manuscript. JSM assisted in study design and direction. KVM designed the laboratory experiments, performed tiRNA sponge and mimic experiments and wrote the manuscript. All authors read and approved the final manuscript.Table S1. Small RNA (sRNA), CCCTC-binding factor (CTCF) chromatin immunoprecipitation (ChIP)-seq and RNA polymerase II (RNAPII) ChIP-seq datasets. ‡Data obtained from ENCODE/UCSC and can be retrieved from the publicly available ENCODE UCSC genome browser (http://genome.ucsc.edu/ENCODE/) [http://www.ncbi.nlm.nih.gov/geo/). MCF-7 CTCF ChIP-seq data can be found in the EBI Array Express Archive (http://www.ebi.ac.uk/arrayexpress/).ENCODE/) ,9. *All Click here for fileFigure S1. Enrichment of transcription initiation (ti)RNAs at sites of conserved CCCTC-binding factor (CTCF) binding genome-wide. CTCF sites were obtained from the ENCODE Broad Institute UCSC Histone Mods tracks. Only those with significant peaks conserved across GM12878, HepG2, HMEC, HSMM, HUVEC, K562, NHEK and NHLF cell lines were considered in this analysis. (a) Enrichment of small RNAs at all conserved CTCF sites (gray), and conserved CTCF sites with evidence of conserved RNA polymerase II (RNAPII) binding in HUVEC, K562 and NHEK cells (black). (b-d) The size distribution of small RNAs found at conserved CTCF sites with evidence of RNAPII binding in (b) THP-1, (c) 5-8f and (d) MCF cells. In (b) and (c) nuclear small RNAs are shown in black, and cytoplasmic small RNAs are depicted in gray.Click here for fileTable S2. Conserved CCCTC-binding factor (CTCF) and CTCF-RNA polymerase II (RNAPII) site intersection with small RNAs (sRNAs).Click here for fileFigure S2. Enrichment of transcription initiation (ti)RNAs at sites of subsets of conserved CCCTC-binding factor (CTCF) binding sites genome-wide. CTCF sites were parsed to (i) exclude any that mapped within 500 bp of a TSSs or overlapped repeat masker, small RNA or Ensembl gene annotations less than 300 nucleotides, (ii) into groups by cell type where 'cancer' was derived from MCF-7, K562 and HepG2 data and normal was derived from GM12878, HUVEC, HMEC, HSMM, NHEK and NHLF data, and (iii) intersected with the most robust RNA polymerase II (RNAPII) data for each group . Note that tiRNAs are highly enriched at CTCF-RNAPII sites even in these highly reduced sets.Click here for fileFigure S3. The intronic p21  CCCTC-binding factor-RNA polymerase II (CTCF-RNAPII) site is highly conserved in mammals. In the top panel the p21/CDKN1A locus in mouse is shown. The bottom panel is a focused view of the intronic CTCF-RNAPII site. Note the conserved CTCF site, RNAPII binding, and high conservation of the site itself and the sequence immediately to the left (5' with respect to p21), which is the site of transcription initiation (ti)RNA biogenesis in humans.Click here for fileFigure S4. p21  CCCTC-binding factor (CTCF) proximal (cp)-transcription initiation (ti)RNA RNA sponge and mimics effects are conserved in THP-1 cells. Samples were prepared and analyzed identical to those shown in Figure p21 mRNA levels in response to the cp-tiRNA sponge, and the decrease in expression in response to cp-tiRNA mimics.Click here for fileFigure S5. A schematic of the CCCTC-binding factor (CTCF) sites proximal to C2orf42 and StAR-related lipid transfer domain containing 13 (STARD13).  Gene models are shown at the top of each panel, followed by the collapsed density of small RNAs in three datasets, CTCF binding density in nine human cell types, and RNA polymerase II (RNAPII) binding in three human cell lines. In (a) the conserved C2orf42 CTCF site is approximately 5 kb downstream of the 3' untranslated region (UTR). The CTCF site in (b) sits approximately halfway between STARD13 and RFC3.Click here for fileFigure S6. The effect of the CCCTC-binding factor (CTCF) proximal (cp)-transcription initiation (ti)RNA RNA sponge on CTCF sites proximal to C2orf42. (a) The effects of the C2Orf42 sponge on CTCF localization. (b) The effects of C2Orf41 tiRNA sponges on mRNA expression. Experiments were standardized to pCDNA transfected MCF-7 cells. (c) The effects of the C2Orf41 sponge on histone H3 localization. Samples were analyzed as indicated 72 h post transfection. The averages of triplicate transfected samples are shown with the error bars representative of the standard errors of the means, and P values from paired t tests.  No antibody values were subtracted from each IP, and the resultant values were standardized to the input for each sample.Click here for fileTable S3. Oligonucleotides used for cloning, quantitative reverse transcription (qRT)-PCR, and chromatin immunoprecipitation (ChIP) analysis.Click here for file


In this study, we examined the functional significance of miR-18a in CRC.in silico search and confirmed by luciferase activity assay and Western blot. DNA damage was measured by comet assay. Gene function was measured by cell viability, colony formation and apoptosis assays.Expression of miR-18a was investigated in 45 CRC patients. Potential target genes of miR-18a were predicted by (p<0.0001). Through in silico search, the 3′UTR of Ataxia telangiectasia mutated (ATM) contains a conserved miR-18a binding site. Expression of ATM was down-regulated in CRC tumors (p<0.0001) and inversely correlated with miR-18a expression . Over-expression of miR-18a in colon cancer cells significantly reduced the luciferase activity of the construct with wild-type ATM 3′UTR but not that with mutant ATM 3′UTR, inferring a direct interaction of miR-18a with ATM 3′UTR. This was further confirmed by the down-regulation of ATM protein by miR-18a. As ATM is a key enzyme in DNA damage repair, we evaluated the effect of miR-18a on DNA double-strand breaks. Ectopic expression of miR-18a significantly inhibited the repair of DNA damage induced by etoposide (p<0.001), leading to accumulation of DNA damage, increase in cell apoptosis and poor clonogenic survival.The up-regulation of miR-18a was validated and confirmed in 45 primary CRC tumors compared with adjacent normal tissues miR-18a attenuates cellular repair of DNA double-strand breaks by directly suppressing ATM, a key enzyme in DNA damage repair. They exert the effects by targeting the RNA-induced silencing complex (RISC) to complementary sites in the 3′ untranslated region (UTR) of their target genes miR-18a belongs to the miR-17-92 cluster, which is located at chromosome 13q31.1 region. The oncogenic role of the miR-17-92 cluster is well documented. Over-expression of the cluster is associated with accelerated tumor growth Rectal tumor and adjacent non-tumorous tissues were obtained from 45 patients with histologically-confirmed rectal cancer when they underwent surgery at the Prince of Wales Hospital, Hong Kong during 1999 to 2003. Normal rectal mucosa was obtained from healthy controls during colonoscopy at the Prince of Wales Hospital during 2009. All subjects provided their written informed consent prior to specimen collection. The study protocol and consent procedure were approved by the Ethics Committee of The Chinese University of Hong Kong.in vitro assays because these two cell lines express functional ATM in response to DNA double-strand break (DSBs) 2 at 37°C. The precursor of miR-18a (pre-miR-18a) and negative control (pre-miR-ctrl) were purchased from Applied Biosystems . Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to manufacturer’s guide.CRC cell lines HCT-116 and HT-29 were adopted for www.targetscan.org) and miRanda (www.microRNA.org). Sequences with the wild-type or mutant seed regions were cloned into pMIR-REPORT luciferase vector (Applied Biosystems). The mutant ATM 3′UTR sequence was prepared by mutating 5 nucleotides in the seed region. The synthesized oligos were shown as follows:The potential miR-18a binding site in ATM 3′ untranslation region (3′UTR) was predicted by targetscan (Wild-type sense strand:5′-CTAGTTGTGTCCCAATTTCAAGTATTTTAATTGCACCTTAATGAAATTATCGAGCT-3′.Wild-type anti-sense strand:5′-CGATAATTTCATTAAGGTGCAATTAAAATACTTGAAATTGGGACACAA-3′.Mutant sense strand:5′-CTAGTTGTGTCCCAATTTCAAGTATTTTAATTTCGTATCAATGAAATTATCGAGCT-3′.Mutant anti-sense strand:5′-CGATAATTTCATTGATACGAAATTAAAATACTTGAAATTGGGACACAA-3′.Renilla luciferase report vector (195 ng/well) and Firefly luciferase vector (5 ng/well) using Lipofectamine 2000 (Invitrogen). Cells were harvested 48 hours posttransfection and luciferase activities were analyzed by the dual-luciferase reporter assay system .The cell lines transiently transfected with pre-miR-18a or pre-miR negative control  in 24-well plates were co-transfected with Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of individual miRNA was performed using the TaqMan miRNA reverse transcription kit (Applied Biosystems) and the TaqMan human miRNA assay  based on a modified protocol from Applied Biosystems 5′- GGAGAGCTGGAAAGCATTGG-3′; Reverse: 5′- TGAGAAGCTGGGAGTGTTTCTG-3′. GAPDH: Forward: 5′-GAAGGTGAAGGTCGGAGT-3′ Reverse: 5′-GAAGATGGTGATGGGATTTC-3′.For ATM mRNA quantitation, total RNA was reverse transcribed with random primer using Reverse Transcription Kit (Applied Biosystems), and real-time PCR was set with Power SYBR Green PCR Master Mix (Applied Biosystems). ATM expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Primer sequences are as follows: ATM: Forward: Total protein was extracted and protein concentration was measured by the Bradford DC protein assay . 20 to 40 µl of protein from each sample were separated on 8% Bis/Tris-polyacrylamide gel through electrophoresis and blotted onto nitrocellulose membranes . Blots were immunostained with primary antibodies at 4°C overnight and secondary antibody at room temperature for 1 hour. Anti-ATM (2C1) antibody was purchased from Genetex . Anti-phospho-Checkpoint kinase 2 antibody (Thr68) was purchased from Cell Signaling . Anti-GAPDH (SC-25778) antibody was purchased from Santa Cruz Biotechnology .HCT-116 cells were transiently transfected with pre-miR-18a or pre-miR negative control  in 24-well plates. After one hour of incubation with 2 µM etoposide or DMSO, the cells were either harvested or switched to medium without etoposide for 2 hours to allow DNA damage repair. At the end of the experiment, cells were harvested for comet assay according to the manufacturer’s guide . The tail moments of the cell comets were analysed automatically using a comet analysis software, CometScore . Tail moments of 50 or more cells per slide were determined.5 per well) were plated in a 24-well plate and transfected with pre-miR-18a or pre-miR-ctrl at 15 nM. Twenty-four hours after transfection, cells were incubated with 2 µM etoposide or DMSO for 1 hour, collected and seeded (500–1000/well) in a fresh 24-well plate for 9 days. Colonies were counted after staining with Harris hematoxylin solution. Cell viability was determined by the 3--2, 5-diphenyltetrazolium bromide (MTT) assay (Promega) according to manufacturer’s guide. Briefly, MTT solution was added to each well at a final concentration of 1 mg/ml per well and the plates were incubated at 37°C for another 3 h. After incubation, 200 µl of DMSO was added to each well to dissolve the formazan formed and the absorbance was read at 570 nm using a spectrophotometer. All experiments were triplicated.Cells (1×105 per well) were seeded in a 24-well plate and transfected with pre-miR-18a or pre-miR-ctrl at 15 nM. Twenty-four hours after transfection, cells were incubated with 2 µM etoposide or DMSO for 1 hour. Apoptosis was assessed by flow cytometry after staining with Annexin V (FITC-conjugated)  and 7-amino-actinomycin .Cells . Difference in cell growth curves was determined by repeated measures ANOVA. Progression-free survival analysis was done by the Kaplan–Meier method and the results were tested using the Log-rank test. p<0.05 was taken as statistical significance. All statistical tests except regression analysis were done by Graphpad Prism 5.0 . = 0.005, Among the 45 pairs of rectal cancer tissue samples, 44 pairs had higher miR-18a expression in tumor than in adjacent normal tissue . Without the induction of DNA damage, over-expression of miR-18a did not have significant effect in cell growth as compared to cells transfected with precursor control both in HT-29  and in HWithout the induction of DNA damage, over-expression of miR-18a did not induce significant effect in cell apoptosis in HT-29 and in HCT-116 . ExposurIn this study, a clear connection between miR-18a and the gene ATM was established. Luciferase reporter assay and western blot analysis confirmed the interaction, which was through the binding of miR-18a to the 3′ untranslated region of ATM mRNA and subsequently suppressed its protein translation and its activity. This association was most evident from the inverse correlation between miR-18a and ATM in rectum tumor tissues (p<0.01).ATM is a high molecular weight protein kinase that plays a central and early role in promoting repair of DNA DSBs, which are one form of the most cytotoxic DNA lesions that arise through both endogenous (e.g. oxidative stress) and exogenous (e.g. ionizing radiation and genotoxic agents) sources. In unstimulated cell, ATM mostly exists as an inactive homodimer or multimer, with the kinase domain of one ATM protein bound to the internal domain of another ATM protein containing the serine 1981-phosphorylation site Compromised DNA repair mechanism due to loss of ATM function is a known predisposition to various diseases. The most evident example being the inherited autosomal recessive disorder, Ataxia-telangiectasia (A–T), which results from loss of ATM protein expression or functional protein product. The disease is characterized by progressive cerebellar ataxia, neuro-degeneration, radiosensitivity, cell-cycle checkpoint defects, genome instability, and a predisposition to various forms of cancer in vitro data clearly demonstrated the suppression of ATM by miR-18a sensitized cancer cells to etoposide, the role of ATM role on chemo-resistance may vary in a chemotherapeutic-specific and tumor-specific manner in vivo. Besides, miR-18a-associated recurrence can also be mediated through other potential target genes that induce its oncogenic nature in vivo. This hypothesis, however, needs further investigation and validation. The establishment of miR-18a as recurrence marker also needs to be validated in a cohort of larger sample size.Currently, tumor stage is the most important prognostic indicator for CRC patients. Nevertheless, many patients developed recurrence after surgical resection regardless of stage or the provision of adjuvant chemotherapy. Additional prognostic biomarkers are needed to provide better recurrence risk assessment so patients can benefit from close follow-up. We found high miR-18a level is associated with higher recurrence rate and quicker recurrence. It remains to be elucidated whether this phenomenon is mediated through ATM or other miR-18a target genes. Nevertheless, the role of ATM in predicting chemo-/radio-therapy resistance in a clinical setting remains not clearly established. Roossink et al. reported that ATM activation induced protective role to chemo−/radio-therapy in a cohort of cervical cancer patients In conclusion, we identified ATM, a protein crucial to DNA repair, as the target of miR-18a. In rectal cancer tissues, expression of miR-18a and ATM correlated inversely. Ectopic expression miR-18a suppresses ATM expression and attenuates DNA DSB repair. miR-18a, a frequently up-regulated miRNA in CRC, induces its oncogenic effect at least partly through suppressing ATM. Moreover, tumor miR-18a level is a potential marker for rectal cancer recurrence.


Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long β-hairpin (Q51–Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the  Salmonella typhimurium. Employing solid-state NMR, we could determine local structure parameters such as dihedral angles and inter-nuclear proximities for this supramolecular assembly. Concurrently, a high-resolution cryo-electron microscopy density map of the T3SS needle of Shigella flexneri was obtained by Fujii et al. Modeling of the Shigella needle subunit protein to fit the EM density produced a model incompatible with the atomic model of the Salmonella needle in terms of secondary structure and subunit orientation. Here, we determined directly the secondary structure of the Shigella needle subunit using solid-state NMR, and its orientation using in vitro and in vivo immunogold labeling in functional needles. We found that Shigella subunits adopt the same secondary structure and orientation as in the atomic model of Salmonella, and we generated a homology model of the Shigella needle consistent with the EM density. Knowing the common T3SS needle architecture is essential for understanding the secretion mechanism and interactions of the needle with other components of the T3SS, and to develop therapeutics.Gram-negative bacteria use a molecular machinery called the Type Three Secretion System (T3SS) to deliver toxic proteins to the host cell. Our research group has recently solved the structure of the extracellular T3SS needle of  Shigella), intestinal inflammations and diarrhea , melioidosis (Burkholderia), the plague (Yersinia), cholera (Vibrio) and pneumonia (Pseudomonas) Shigella infections, is responsible for 165 million infections and 1.5 million deaths annually The Type Three Secretion System (T3SS) is a secretion machinery used by Gram-negative bacteria to deliver virulence factors into eukaryotic host cells Shigella, PrgI in Salmonella and BsaL in Burkholderia bacteria. Earlier studies of Shigella flexneri T3SS needles by X-ray fiber diffraction and cryo-electron microscopy (cryoEM) showed that the MxiH subunits are disposed in a helical arrangement, with 5.6 subunits per turn and a 24-Å helical pitch CΔ5 by solution-state NMR CΔ5 by X-ray crystallography CΔ5 by solution-state NMR T3SS are formed by the assembly of ∼20–25 different proteins, some of these in multiple copies. They consist of a hollow needle and a basal body which anchors the needle to both bacterial membranes Shigella was modeled by Deane and coworkers CΔ5 crystal monomers into a 16-Å cryoEM density map Shigella MxiH needle was presented by Fujii and coworkers Salmonella typhimurium T3SS needle in vitro and in vivoIn order to gain further insight into the function of Type Three Secretion Systems, it is necessary to obtain an atomic structure of wild-type (WT) needles in their assembled state. The pseudoatomic T3SS needle architecture of S. flexneri T3SS needles can be made and experimentally verified. In the current study, we thus present solid-state NMR data and immunogold labeling results obtained on WT needles of S. flexneri which allow us to address the discrepancy between the two models and establish the common architecture of T3SS needles.Although the two proposed models show fundamental differences in their topology, MxiH and PrgI present a high sequence identity >60%, . From thin vitro preparation of functional Salmonella T3SS needles Shigella T3SS needles (13C cross-polarization spectra (13C line-widths measured for [1-13C]glucose (Glc)- and [2-13C]Glc-labeled needles range from 0.09 to 0.25 ppm (between 20 and 55 Hz on an 850 MHz spectrometer), on par with other protein samples showing the highest resolution in solid-state NMR: microcrystalline GB1 S. typhimurium PrgI T3SS needles The protocol developed for the  needles A. The n spectra 3B revea13C and 15N chemical shifts -13C experiments. Due to the high spectral resolution of the sample, most of the assignment could be carried out on uniformly-labeled ([U-13C6]Glc) needles using 2D spectroscopy. An example of the spectral resolution obtained in [U-13C6]Glc needles is presented in a 13C-13C proton-driven spin diffusion (PDSD) spectrum  T3SS needles. In sparsely-labeled samples, the dilution of 13C spins results in an improved 13C resolution due to the removal of one-bond 13C-13C dipolar and JCC couplings, and in the simplification of spectra 13C]Glc- and [2-13C]Glc labeling patterns The solid-state NMR assignment of 1H-13C spectrum, all residues in the MxiH subunit protein exhibit a rigid conformation.Residues extending from S2 to R83 in the MxiH sequence are all observed in cross-polarization based solid-state NMR spectra, indicating a high structural rigidity. Consequently, with the exception of Met1 detected in an INEPT CΔ5 (PDB ID: 2CA5) where two distinct conformations of MxiH are present in the unit cell No peak doubling could be observed in solid-state NMR spectra , denotinAs backbone chemical shifts are strongly correlated to secondary structure et al. are not compatible with the experimental chemical shifts of MxiH , and needles were polymerized. The tag is recognized by an Anti-His tag antibody which is imaged using protein A Gold (mxiH-N(His) needles incubated without the Anti-His tag antibody , (mxiH-C(Strep), (mxiH and mxiH-N(Strep) could assemble secretion-competent needles, as shown in a protein secretion assay (mxiH-C(Strep) did not assemble any needle (mxiH-N(Strep) formed needles decorated with gold particles. These results show that the MxiH subunit N-terminus is exposed at the surface of the needle and corroborate that MxiH needles adopt the topology found in the PrgI needle solid-state NMR structure In order to determine the placement of the N- and the C-terminus in the MxiH needle, immunogold labeling experiments were performed both n A Gold B. Immunn A Gold A and foantibody C, exclu WT mxiH E, an N-(Strep), F or a C(Strep), G. The son assay D, but ty needle G and waYersinia pestis, YscF, which shows lower sequence identity to MxiH (25%). Sequences for EscF proteins are more evolutionary distant and were not included in any of the clusters. They also lack the canonical P-(S/D)-(D/N)-P loop motif and the variable extension.As T3SS are employed by a variety of bacterial pathogens The loop and C-terminal region contain a large number of residues, 11, that are highly conserved in both clusters, compared to only two residues in the N-terminal region. Those highly conserved residues, indicated with stars in S. flexneri needle assemblies were prepared by in vitro polymerization and studied by solid-state NMR. The MxiH needle subunit is predominantly rigid (S2-R83). Our chemical shift assignment  or 8 (molecule B) C-terminal residues In the current study, WT Shigella MxiH and Salmonella PrgI subunits in their needle assembled state is highly similar, as revealed by the comparison of secondary chemical shifts , followed by the N-terminal α-helix (L12-A38) with a kink at T23, a loop (E39-P44) and the C-terminal α-helix (Q45-R83). The conformation of l shifts C and 4Det al. is that the measurements were carried out on S. flexneri serotype 6 (this study) and serotype 2. Compared to serotype 6, the sequence of serotype 2 has the following mutations: D7N, K8D, Q29E, S32L, N35D, A36K and E39K. Since no amino acid substitutions between the two serotypes are located in the conjectured β-hairpin region of MxiH (Q51–Q64), rearrangement of the secondary structure of the C-terminal helix by these mutations would likely involve even greater alterations locally at the site of the mutation, which are not observed. The finding that PrgI sustains a significantly greater number of N-terminal substitutions while preserving a C-terminal conformation highly similar to MxiH further discredits this hypothesis. We conclude that the incompatibility between this model and the experimental data results from remodeling of the monomeric subunit structure to fit the cryoEM density map without the guidance of site-specific local structural information such as experimentally-determined dihedral angles and distance restraints.In contrast, the chemical shifts observed in this work are incompatible with the secondary structure identified in the previous MxiH needle model S. typhimuriumS. flexneri needles which accounts for all available experimental data, including the 7.7-Å cryoEM density map. Although the correlation to the cryoEM density is higher for the previous model by Fujii et al. (0.72) compared to our PrgI-based homology model (0.66), this is expected as no EM bias was used in the building of the current homology model. The previous model also has significantly higher full-atom energy after Rosetta refinement , D-[1-13C]glucose or D-[2-13C]glucose as carbon source.MxiH was expressed and purified following the protocol established for 1H frequencies on Avance I and Avance III spectrometers  at a spinning rate of 11 kHz and a temperature of 5.5°C. Additional experimental details can be found in supplementary Table S1 in MxiH needles were ultra-centrifuged and transferred into 4.0-mm magic-angle spinning rotors. Solid-state NMR experiments were conducted at 600 MHz, 800 MHz and 850 MHz Shigella mxiH-knockout cells (ΔmxiH) were complemented with either wild-type mxiH, mxiH with N-terminal Strep-tag or mxiH with a C-terminal Strep-tag in pASK-IBA vectors (IBA lifesciences) and induced with 0.2 µg/ml anhydrotetracycline for 1 hr. The cells were fixed in 2% paraformaldehyde and immunogold labeled using 1∶40 diluted StrepMAB-Classic (IBA lifesciences) for 1 hr followed by 1∶40 diluted 12-nm gold-conjugated secondary antibody for 1 hr. Protein secretion assays were performed by addition of 20 µg/ml CR followed by western blot analysis against IpaB, IpaC and DnaK.Recombinantly-produced MxiH needles were labeled using an Anti-penta-Histidine tag monoclonal antibody (Invitrogen). The labeling was performed using a 1∶200 diluted antibody and 10-nm protein A Gold . Non-polar Sequences of commonly studied T3SS needle proteins were aligned using Clustal W version 2 et al. was done in the following manner: the full 29-subunit assembly was generated from the coordinates of the monomeric subunit using the symmetry-related operations described in the header of the PDB file (PDB ID: 3J0W), followed by the aforementioned symmetric refinement in Rosetta. The refinement led only to minor changes relative to the starting coordinates . 100 models were generated in this manner and the median energy of the 10 lowest-energy models was reported  under accession number 18651.The Text S1Supporting information. Figure S1, solid-state NMR 13C-13C spectrum of MxiH needles. Figure S2, back-prediction of secondary chemical shifts. Table S1, list of 2D solid-state NMR experiments. Table S2, chemical shifts of Shigella flexneri MxiH needles. Table S3, source of primary sequences for multiple sequence alignment.(DOC)Click here for additional data file.


In this study, we constructed EGSs from a variant to target the overlapping region of the S mRNA, pre-S/L mRNA, and pregenomic RNA (pgRNA) of hepatitis B virus (HBV), which are essential for viral replication and infection. The EGS variant was about 50-fold more efficient in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Following Salmonella-mediated gene delivery, the EGSs were expressed in cultured HBV-carrying cells. A reduction of about 97% and 75% in the level of HBV RNAs and proteins and an inhibition of about 6,000- and 130-fold in the levels of capsid-associated HBV DNA were observed in cells treated with Salmonella vectors carrying the expression cassette for the variant and the tRNA-derived EGS, respectively. Our study provides direct evidence that the EGS variant is more effective in blocking HBV gene expression and DNA replication than the tRNA-derived EGS. Furthermore, these results demonstrate the feasibility of developing Salmonella-mediated gene delivery of highly active EGS RNA variants as a novel approach for gene-targeting applications such as anti-HBV therapy. External guide sequences (EGSs) are RNA molecules that consist of a sequence complementary to a target mRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, for specific degradation of the target mRNA. We have previously used an  Primer extension experiments were carried out by incubation of 8 µL of a mixture of total cellular RNA (10 µg) and 50,000 cpm of primers, 4 µL 5× RT buffer, 2 µL 10 mM dATP, 2 µL 10 mM dTTP, 2 µL 10 mM dGTP, 2 µL 10 mM dCTP, 0.5 µL RNasin, and 1 µL AMV reverse transcriptase . After incubation at 42°C for 2 h, the reaction products were extracted with phenol chloroform, then precipitated by cold ethanol, and finally separated in 8% denaturing gels. The sites that blocked the primer extension reaction by reverse transcription, which were revealed by analyzing the gels using a STORM840 Phosphorimager, represented the potential locations modified by DMS -labeled s38 with human RNase P at 37°C in buffer A . Cleavage products were separated in denaturing gels and analyzed with a STORM840 phosphorimager . Assays to determine kinetic parameters were performed under multiple turnover conditions, as described previously -radiolabeled DNA probes that contained the DNA sequences coding for S-C386 and S-SER, and finally analyzed with a STORM840 phosphorimager  -radiolabeled DNA probes that contained the HBV DNA sequence and human H1 RNA, and analyzed with a phosphorimager. The DNA probes used to detect HBV RNAs and human H1 RNA were synthesized from plasmids pHBV1.3 and pH1 RNA, respectively  for the cleavage reactions. S-C386 was highly efficient in directing human RNase P to cleave s38 and was at least 50-fold more efficient than S-SER  using gel-shift assays for separating substrate-EGS complexes in non-denaturing polyacrylamide gels. S-C386 exhibited about 70 times higher binding affinity to s38 than S-SER  to the target S RNA sequence may be better than that of the EGS (i.e. S-SER) derived from the natural tRNA sequence. The binding affinities of EGS S-C386 and S-SER to substrate s38 were determined by measuring the dissociation constant (K3-fold slower than that in the presence of S-C386 (in vitro (data not shown).Three control EGSs were also used in our study. S-C386-C and S-SER-C were derived from S-C386 and S-SER, respectively, and contained base substitutions (5’-UUC-3’ -> AAG) at the three highly conserved positions in the T-loop of these EGSs , which is a major virulence and proinflammatory factor expressed on the surface of Salmonella  in directing RNase P to cleave the S mRNA sequence in vitro than S-SER, an EGS derived from the natural tRNASer sequence. Moreover, S-C386 inhibited HBV gene expression (i.e. HBV transcripts and HBsAg/HBeAg) in cultured cells by about 97-98% and was more effective than S-SER, which reduced HBV gene expression by about 75%. In contrast, a reduction of less than 10% in HBV gene expression and DNA replication was observed in cells that expressed control EGS S-C386-C, S-SER-C, or TK112. S-C386-C and S-SER-C exhibited similar binding affinity to s38 as S-C386 and S-SER, respectively, but were inactive in directing RNase P-mediated cleavage due to the presence of the mutations at the T-loop that precluded RNase P recognition (max(apparent) /Km(apparent)] in inducing RNase P to cleave the S mRNA sequence in vitro is also more effective in inhibiting HBV gene expression and DNA replication in cultured cells and that increasing the activity of EGS in directing RNase P cleavage in vitro may lead to improved efficacy in inhibiting gene expression in cultured cells.The EGS-based technology represents an attractive approach for gene inactivation since it utilizes endogenous RNase P to generate highly efficient and specific cleavage of the target RNA ,40. HoweSalmonella strains with better gene delivery activity, should facilitate the development of the EGS-based technology as a promising gene targeting approach for in vivo applications.Our results also suggest that the increased targeting activity of EGS S-C386 may be due to the enhanced stability of the mRNA-EGS complexes formed between S-C386 and the target HBV RNA sequence. S-C386 bound to substrate s38 with at least 70-fold greater affinity than S-SER , and E. Salmonella-mediated gene delivery are active and block HBV replication in cultured cells. First, targeted gene transfer of the EGS constructs by Salmonella yields substantial expression of the EGSs  is a major human pathogen chronically infecting more than 400 million individuals worldwide . Hepatoc 1 month ,49. MoreToday, there is no definitive cure for chronic HBV infection. The current FDA-approved anti-HBV medications for the management of chronic HBV infections include alpha interferon, and five nucleoside analogues  that act as potent inhibitors of viral polymerase but rarely cure HBV infection ,47. The


Therefore, we investigated whether G0S2 plays a role in hepatic lipid metabolism. Adenovirus-mediated expression of G0S2 (Ad-G0S2) potently induced fatty liver in mice. The liver mass of Ad-G0S2-infected mice was markedly increased with excess triglyceride content compared to the control mice. G0S2 did not change cellular cholesterol levels in hepatocytes. G0S2 was found to be co-localized with adipose triglyceride lipase at the surface of lipid droplets. Hepatic G0S2 overexpression resulted in an increase in plasma Low-density lipoprotein (LDL)/Very-Low-density (VLDL) lipoprotein cholesterol level. Plasma High-density lipoprotein (HDL) cholesterol and ketone body levels were slightly decreased in Ad-G0S2 injected mice. G0S2 also increased the accumulation of neutral lipids in cultured HepG2 and L02 cells. However, G0S2 overexpression in the liver significantly improved glucose tolerance in mice. Livers expressing G0S2 exhibited increased 6-(N-(7-nitrobenz-2-oxa-1-3-diazol-4-yl) amino)-6-deoxyglucose uptake compared with livers transfected with control adenovirus. Taken together, our results provide evidence supporting an important role for G0S2 as a regulator of triglyceride content in the liver and suggest that G0S2 may be a molecular target for the treatment of insulin resistance and other obesity-related metabolic disorders.Nonalcoholic fatty liver disease is associated with obesity and insulin resistance. Factors that regulate the disposal of hepatic triglycerides contribute to the development of hepatic steatosis. G Hepatic steatosis, also called fatty liver, is caused by the abnormal retention of triglycerides and other fats within liver cells. Obesity, diabetes, and excessive alcohol consumption may contribute to hepatic steatosis –3; more Within the liver, triglycerides accumulate in lipid droplets, and their deposition is dependent on their biosynthesis and elimination rates . A decre0/G1 switch gene 2 (G0S2) was initially found to be differentially expressed in lymphocytes during the lectin-induced switch from the G0 to G1 phase of the cell cycle [Gll cycle ,11. Bothll cycle . The actll cycle , PPAR-β/ll cycle , and retll cycle ,15 as well cycle , suggestll cycle . ATGL is2 at 37° C. DMEM and FBS were purchased from Invitrogen . Antibodies against G0S2 (N-13), ATGL (F-7), and horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology . FITC-conjugated donkey anti-rabbit and TRITC-conjugated goat anti-mouse antibodies were from Proteintech Group, Inc. .HepG2, a hepatocytoma cell line, and L02, a normal hepatic cell line , were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine in 5% COAn adenovirus (Ad) expressing G0S2 was constructed using Adeno-X Expression System 2 following the manufacturer’s instruction . Briefly, the full-length coding region of G0S2 was subcloned into the pDNR-CMV vector and transferred to the pLP-Adeno-X-CMV Acceptor Vector via Cre-loxP-mediated recombination. The Ad was amplified and purified using the cesium chloride method. Cells were infected with the Ad at a multiplicity of infection (MOI) of 100.We used the pAc-GFP-C1 vector to construct plasmids for the eukaryotic expression of the full-length coding region of the human G0S2 gene. All plasmids were constructed using an In-Fusion™ Dry-Down PCR Cloning Kit according to the manufacturer’s protocol . Plasmids were purified using the Promega PureYield Plasmid System. Cultured cells were transfected with the Ac-GFP-G0S2 plasmid using the Lipofectamine™ LTX Reagent  and incubated for 24 h. Immunofluorescence was visualized using a laser scanning confocal microscope .9 pfu) in 100 µL sterilized phosphate-buffered saline (PBS) was delivered by tail vein injection. Diabetes was induced by multiple subdiabetogenic intraperitoneal (i.p.) injections of streptozotocin (STZ) freshly dissolved in 10 mM Na-citrate buffer (pH 4.5) (50 mg STZ/kg body weight daily for 5 consecutive days) . The experimental group of mice had food withdrawn for 16 h, with ad libitum access to water, previous to sacrifice. Liver tissue was removed for the measurements of organ weight and triglyceride content, RNA and protein extraction, and histological examinations.All experiments using mice were conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and approved by the Animal Care and Use Committee of Hubei University of Medicine. Male 4-week-old Swiss mice were used in all experiments. The animals were fed with standard rodent chow and maintained on a 12-h artificial light-dark cycle. Ad-LacZ or Ad-G0S2  was injected into each mouse i.p. (0.2 U/kg body weight). Blood glucose levels were measured using a glucose meter at 15, 30, 60, and 90 min after the insulin challenge.Freshly dissected liver was fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose in PBS for another day, and then frozen in OCT blocks. Cryostat sections 7 µm thick were processed for Oil Red O staining; thereafter, the slices were fractionated with 60% isopropanol for 1 min and washed with PBS for 2 min. To measure the cellular accumulation of neutral lipid droplets, HepG2 and L02 cells were fixed in 4% paraformaldehyde and stained with Oil Red O solution for 30 min at room temperature. The cells were washed with 60% isopropanol. After staining, the cells were washed with PBS to remove unbound dye. To quantify Oil Red O content, the samples were treated with isopropanol and read using a spectrophotometer at 510 nm.Triglyceride and cholesterol levels in the liver and cells were measured using colorimetric kits from Abcam  according to the manufacturer’s instructions. Blood for the determination of lipid parameters was obtained from fasted mice. High-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL)/very-low-density (VLDL) lipoprotein cholesterol, and ketone bodies in serum samples were evaluated, respectively, using colorimetric kits from BioAssay Systems  and a β-Hydroxybutyrate Colorimetric Assay Kit from Abcam  according to the manufacturer’s instruction. A Free Fatty Acid Quantification Kit was purchased from BioVision .® Co-Immunoprecipitation Kit  and then subjected to western blot analysis with the anti-G0S2 antibody. Equal amounts of total protein (30 µg) were also resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes for western blot analysis. The membranes were probed with a primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. The immunocomplexes were visualized with Enhanced Chemiluminescence Plus .For the co-immunoprecipitation assays, 300 µg of total protein extract from mouse livers were immunoprecipitated with the anti-ATGL antibody or pre-immune IgG by using a Dynabeadsin vivo. A fluorescent d-glucose analog, 6- amino)-6-deoxyglucose , was used to detect glucose uptake by the liver. LacZ- or G0S2-infected mice were fasted overnight and then a glucose solution (2 g/kg) was administered orally. Fifty minutes later, 100 µL of 2 mM 6-NBDG was injected intravenously via the tail vein into each mouse. Another 15 min later, the animals were perfused with PBS via the left ventricle. The liver was minced and homogenized in a lysis buffer supplemented with a protease inhibitor cocktail. The soluble fraction was obtained by centrifugation. The 6-NBDG content in equal amounts of protein lysate was measured by fluorescence intensity (excitation/emission of ~465/540 nm).Glucose uptake by hepatic cells was assessed P-values less than 0.05 were considered statistically significant.Data are expressed as the mean ± standard error of the mean (SEM). Differences were assessed by analysis of the variance or Student’s t-test. in vivo hepatic function of G0S2, a recombinant Ad expressing G0S2 was generated. The mice were injected via the tail vein with the Ad vectors. The expression of G0S2 protein in the liver and fat tissues were verified by western blot analysis  . The Ad-Systemic free fatty acids are the major substrate for VLDL-TAG and ketone body 3-hydroxybutyrate production. HDL cholesterol and LDL cholesterol are two major components of cholesterol. We first examined the effect of G0S2 overexpression on HDL cholesterol and LDL/VLDL cholesterol levels in plasma. The resulting data showed that G0S2 overexpression increased LDL/VLDL cholesterol levels, while it slightly decreased HDL cholesterol levels in plasma . HoweverSince G0S2 functionally associates with ATGL and regulates its action in adipocytes, we examined whether G0S2 physically interacts with ATGL in the mouse liver. The subcellular localization of G0S2 and ATGL was determined by using immunofluorescence staining. LDs in the liver were visualized under a phase-contrast microscope and confirmed using Oil Red O staining. G0S2 was found to be localized at the surface of lipid droplets and co-localized with ATGL . To confLipolysis of cellular triglycerides by ATGL generates mediators involved in the generation of lipid ligands for PPAR activation. ATGL deficiency in mice can decrease the mRNA levels of PPAR-α and PPAR-δ target genes in cardiomyocytes. We examined the expression of aP2, Cyp4a10, Ehhadh, Fgf21, and Aldh3a2 mRNA in the liver. The resulting data showed that the mRNA levels of Ehhadh and Aldh3a2 in the liver were reduced in the Ad-G0S2-infected mice compared to the control mice. G0S2 overexpression did not alter aP2 or Cyp4a10 expression . We furtin vivo. We found that overexpression of G0S2 in the liver significantly improved glucose tolerance (P < 0.05 at 30 and 60 min) at days 4 . Reportedly, fatty acid oxidation was increased by ATGL overexpression and decreased by ATGL knockdown . The curEndogenous ATGL is localized to the external surface of lipid droplets in adipocytes, and it plays a key role in lipid droplet/adiposome degradation in mammalian cells. G0S2 couples with ATGL and prevents ATGL-mediated basal lipid droplet degradation in adipocytes and HeLa cells . Recent in vivo, but to a lesser extent (1.5-fold) in hepatocytes in vitro. Lipid droplets of Ad-G0S2-infected livers in vivo were also significantly larger than those seen in Ad-G0S2-infected HepG2 and L02 cells in vitro. To address this discrepancy, physiological concentrations of oleic acid were added to the culture media; however, G0S2-induced hepatic lipid accumulation in hepatocytes in vitro was still much weaker than in vivo (data not shown). Furthermore, a high MOI still could not increase lipid accumulation in hepatocytes. The differences between the in vitro and in vivo results may indicate that internal conditions can modulate G0S2 activity and the global metabolic effects of G0S2 may also contribute to the development of hepatic steatosis.It is worth noting that G0S2-induced hepatic steatosis was very potent (by 3-fold) in vivo. Discrepant effects on insulin sensitivity and hepatic steatosis have been observed in several recent studies. Reportedly, liver-specific Akt activation [A number of studies have documented a strong relationship between hepatic steatosis and insulin resistance. Hepatic steatosis is thought to be part of metabolic syndrome ,30. G0S2tivation , angiopotivation , and inhtivation  improvedtivation . Previoutivation . Furthertivation . Hepatictivation . Recentltivation . Thus, GIn summary, our present study provided experimental evidence supporting an important role for G0S2 as a regulator of triglyceride content in the liver. Further investigations are needed to address the functions of G0S2 in the pathophysiology of obesity-related hepatic steatosis in humans.


Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels.The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. Here we present the results of a genetic screen in  Drosophila that was designed to identify regulators of cellular insulin sensitivity in an in vivo context. Surprisingly, we discovered cross-talk between the epidermal growth factor receptor (EGFR)–activated MAPK/ERK and insulin signaling pathways. This regulatory mechanism, which involves transcriptional control of insulin-like receptor gene, is utilized in vivo to maintain circulating glucose at appropriate levels. We provide evidence for a regulatory feed-forward mechanism that allows for dynamic transient responsiveness as well as more stable, long-lasting modulation of insulin responsiveness by growth factor receptor signaling. The combination of these mechanisms may contribute to robustness, allowing metabolism to be appropriately responsive to physiological inputs while mitigating the effects of biological noise.Insulin signaling is an important and conserved physiological regulator of growth, metabolism, and longevity in multicellular animals. Disturbance in insulin signaling is common in human metabolic disorders. For example insulin resistance is a hallmark of diabetes and metabolic syndrome. While the core components of the insulin signaling pathway have been well established, the mechanisms that adjust the insulin responsiveness are only known to a limited extent. Here we present results from a genetic screen in  Drosophila has a single insulin-like receptor protein (InR), which is activated by a family of insulin-like peptides (ILPs) and mediates physiological responses related to both metabolism and growth , through their respective receptors . Therefore, it is perhaps not surprising that cellular insulin sensitivity is modulated by input from other signaling pathways. For example, TORC1-regulated S6 kinase (S6K) inhibits expression of the IRS scaffold proteins, which are recruited to activated insulin/IGF receptors, thereby making cells more insulin resistant Drosophila eye provides such a system. Because insulin signaling limits FOXO activity, overexpression of FOXO can challenge the regulatory capacity of the insulin pathway, creating a sensitized genetic background in vivo modulators of insulin pathway activity, we have uncovered a novel regulatory mechanism influencing insulin sensitivity. We show that the extracellular signal-regulated kinase (ERK)/MAP kinase signaling pathway (for review see insulin-like receptor (inr) gene. This transcriptional regulation is mediated through the Ets-1 orthologue, Pointed, a transcription factor regulated by the MAPK/ERK pathway While regulation of insulin sensitivity is clearly of physiological importance, identifying novel regulatory mechanisms through genetic screens has been challenging due to the need for a sensitive readout that is also amenable to large-scale screening. The PI3K gene, which serves as a positive control. The screen also identified kinase suppressor of ras (ksr) as an enhancer of the FOXO gain-of-function phenotype. Downregulation of ksr by RNAi enhanced the FOXO phenotype, but on its own, did not reduce eye size  encodes an ETS-1 transcription factor that is activated by MAPK/ERK through phosphorylation of a conserved threonine residue, T151 inr expression by depletion of Pointed from S2 cells by RNAi. This caused a prominent reduction of inr mRNA (A/C/G)GGA(A/T)(A/G)-3′; inr gene expression. Reciprocally, Pointed depletion in S2 cells led to a decrease in the level of InR protein  signaling by expression of a dominant negative form of EGFR (dnEGFR) led to downregulation of inr mRNA BSC678, which fully removes the inr gene Exel6186, Df(3R)ED6076 and Df(3R)BSC678 flies were obtained from the Bloomington Stock Center. UAS-RNAi-PI3K, UAS-RNAi-KSR and UAS-RNAi-InR lines were from the Vienna Drosophila RNAi center. pUAST-FOXO-GFP flies were provided by Aurelio Teleman. pUAST-dnEGFR flies were provided by Pernille Rørth. T5pnt flies were provided by Christian Klämbt.PI3K, nucleotides 358–857 of Pi3K92E coding sequence (FBpp0083348); ksr, nt 2224–2710 (FBpp0078413); D-MEK, nt 961–1191 of the ORF plus the first 83 nt of the 3′UTR (FBtr0071313); Raf, nt 522–912 (FBpp0110324);GAP1, nt 153–646 (FBpp0076096); pnt, nt 1541–1957 ; GFP, nt 17–633 of EGFP2, was used as control. S2 cells were treated with 37 nM dsRNA. Cells were transfected using effectene reagent (QIAGEN) with pMT-GAL4, pUAST-FOXO-GFP or pUAST-Myc-Dp110CAAX, or pMT-GFP-PH or pMT-InR-Flag or pMT-Pnt-P2. 0.7 mM CuSO4 was used to induce FOXO, Dp110, GFP-PH, InR or Pnt expression after transfection. The following primers were used to clone InR-Flag into pMT vector with EcoRI, NotI and XhoI sites by fusion: 5′-GGTACCTACTAGTCCAGTGTGGTGGAATTCATGTTCAATATGCCACGGGGAGTGAC-3′; 5′- TTCGAAGGGCCCTCTAGACTCGAGCGGCCGCTTACTTGTCATCGTCGTCCTTGTAGTCCGCCTCCCTTCCGATGAATCCA-3′; 5′- ACGTTGCGCTCGAGCCAGAGCTCGA-3′ and 5′- TCGAGCTCTGGCTCGAGCGCAACGT-3′. The primers used to clone pntp2 into pMT-Myc by SLIC at EcoRI site were: forward, 5′-AGTGCAACTAAAGGGGAATTCATGGAATTGGCGATTTGTAAAACAG-3′; reverse, 5′- GATAAGCTTCTGCTCGAATTCATCCACATCTTTTTTCTCAATCTTAAG-3′. The primers used to clone the inr gene regulatory region into pGL3-Basic at HindIII and XhoI sites were: forward, 5′- GCGTGCTAGCCCGGGCTCGAGTGAGAGTTTCATGTGTCAGA -3′; reverse, 5′- AAGCTTACTTAGATCGCAGATGTTAATTGCACAGCAAGCTC-3′. The primers used to mutate the predicted Pnt consensus site with QuickChange II XL kit were: forward, 5′-GAGAATGCCGGAGATGAAGACGCGAACGAAGATGAAGTCGATG-3′; reverse, 5′- CATCGACTTCATCTTCGTTCGCGTCTTCATCTCCGGCATTCTC-3′.S2 cells were grown at 25°C in SFM (Gibco) supplemented with L-glutamine. dsRNA was prepared using MegascriptT7 (Ambion) with the following templates: st instar larvae were seeded at 50/vial and reared at 25°C. Wandering 3rd instar larvae were dissected. Tissues were fixed in PBS with 4% paraformaldehyde at room temperature for 20 min. Anti-FOXO antibody For FOXO-GFP localization, live S2 cells were imaged using a Leica SP5 confocal microscope. Images were taken of random fields within 15 min after 10 µg/ml insulin boost for 30 min and scored for GFP localization (scoring was done ‘blind’). For GFP-PH images were taken within 10 min after 10 µg/ml insulin boost for 5 min. The ratio of membrane to cytoplasmic GFP levels was measured as pixel intensity along the white line as indicated in Cells were homogenized in SDS sample buffer, boiled and resolved by SDS-PAGE before transfer to nitrocellulose membranes for antibody labeling. Antibodies to phospho-S505-AKT, AKT, P-InR and Myc were from Cell Signaling Technology. Anti-Kinesin was from Cytoskeleton. Phospho-ERK antibody was from Sigma. Anti-S6K is described in Kinesin mRNA, and rp49 was used as a control. The following primers were used: Kinesin-f, 5′-GCTGGACTTCGGTCGTAGAG-3′; Kinesin-r, 5′- CTTTTCATAGCGTCGCTTCC-3′; rp49-f, 5′- GCTAAGCTGTCGCACAAA-3′; rp49-r, 5′- TCCGGTGGGCAGCATGTG-3′; InR-f, 5′- CTGGTGGTGCTGACAGAGAA-3′; InR-r, 5′- GCAGCTGACAACTGGCATTA-3′; pri-InR-f, 5′- CAAGAGACAGCAACAAAAGG-3′; pri-InR-r, 5′- GCTTGCATGTGTTGGTGAGC-3′; KSR-f, 5′- AGCCGAGCGAAGATTGTAAA-3′; KSR-r, 5′- TCCCGATACATGCCTACACA-3′; pnt-f, 5′- CGATGCGAATGCCTACTACACG-3′; pnt-r, 5′- TGCTGGTGTTGTAGCCTGAAC-3′.Total RNA was extracted from S2 cells using QIAGEN RNeasy Mini Kit and treated with On-Column DNase (QIAGEN RNase-Free DNase Set) at room temperature for 15 min to eliminate genomic DNA contamination. Reverse transcription to synthesize the first strand used oligo-dT primers and Superscript RT-III (Invitrogen). PCR was performed using POWER SYBR GREEN Master Mix (Applied Biosystems) and analyzed on Applied Biosystems 7500 fast real-time PCR system. Results were normalized to st instar larvae were seeded at 50/vial and reared at 25°C. Hemolymph was extracted from wandering stage 3rd instar larvae. 2 µl of pooled hemolymph was diluted with 8 µl Tris buffered saline (pH 6.6) and incubated at 70°C for 5 min before clarification by centrifugation at 20 000×g for 1 min. Glucose was measured in 6 µl supernatant using the GAGO-20 kit (Sigma) and normalized to the same amount of TBS as blank control. For trehalose measurement, supernatant was incubated with 7.5 µg trehalase (Sigma) overnight at 37°C and measured using GAGO-20 kit as well. For glycogen and triglyceride measurements, 3rd instar larvae were homogenized using Sartorius Potter-S tissue homogenizer in water with 0.05% Tween. Supernatant was collected after 5 min of heat inactivation at 70°C and centrifugation at 13000 rpm for 3 min. Glycogen and protein levels were measured using Glycogen assay kit (Bio Vision) and Bio-Rad protein assay reagent, respectively. Triglyceride was measured using Sigma Triglyceride kit. Data were normalized to total protein.Newly hatched 1Figure S1Effect of depleting KSR, AKT or Raf on growth in the wing. UAS RNAi transgenes targeting AKT, KSR or Raf were expressed in the posterior compartment of the wing imaginal discs under control of engrailed-Gal4. Knockdown of MAPK/ERK pathway components lead to undergrowth similar to that caused by suppression of insulin signaling pathway. Upper panels: show photographs of the resulting adult wings. Red dots indicate the border between anterior and posterior compartments. Note the reduced area of the P compartment. Lower panels: higher magnification views showing individual cell size .(TIF)Click here for additional data file.Figure S2ksr gene dosage on the FOXO overexpression phenotype. Upper panel: Photographs of adult eyes. From left to right: GMR-Gal4 alone; GMR-Gal4 with one mutant copy of the ksr gene; GMR-Gal4+UAS-FOXO; GMR-Gal4+UAS-FOXO with one mutant copy of the ksr gene. Lower panel: Plot of total eye area measured in pixels from digital images using Image J. Error bars indicate standard deviation from measurement of at least 5 eyes for each genotype. (*) Student's t-test for removing one copy of ksr in GMR-Gal4+UAS-FOXO eyes p<0.05.Effect of reduced (TIF)Click here for additional data file.Figure S3Representative images showing FOXO localization in S2 cells. Subcellular localization of FOXO-GFP in S2 cells visualized by confocal microscopy. Left panel: (N) predominantly nuclear. Middle panel: (CN) equal levels in cytoplasm and nucleus. Right panel: (C) predominantly cytoplasmic.(TIF)Click here for additional data file.Figure S4KSR acts upstream of PI3K. Immunoblots to visualize the level of AKT and ERK phosphorylation in S2 cells transfected to express the membrane-tethered form of Dp110 (CAAX) compared to control cells transfected with the empty vector. Cells were cultured in serum-free medium and were not stimulated by addition of insulin. Upper to lower: antibody to phosphorylated S505 AKT; antibody to total AKT; Antibody to the phosphorylated form of ERK; antibody to the Myc epitope tag to visualize expression of the Dp110 transgene. Samples were run on the same gel, but intervening lanes have been removed as indicated.(TIF)Click here for additional data file.Figure S5inr cis-regulatory region. Thick black arrow lines indicate transcripts of inr. The five putative cis-regulatory regions analyzed by luciferase assay are shown as horizontal lines below. The thick line indicates the active region, which was used to narrow down to the 0.8 kb element in inr gene is indicated.Schematic representation of the fragments used to locate the (TIF)Click here for additional data file.Figure S6pointed alleles modestly but significantly enhanced the FOXO overexpression phenotype in the eye. (A) Adult eyes expressing GMR-Gal4 and UAS-FOXO without or with one mutant copy of the pointed gene (07825pnt or T5pnt as indicated). (B) Plot of total eye area measured in pixels from digital images using ImageJ. Error bars indicate standard deviation from measurement of 7 eyes for each genotype. Student's t-test: (***) p<0.001.Independent (TIF)Click here for additional data file.Figure S7inr function in vivo.  Adult eyes expressing GMR-Gal4 alone or with UAS-FOXO. Exe/+ indicates flies with one copy of the deletion Df(3R)Exel6186 that partially removes inr locus. The total area of eyes was measured in pixels from digital images using ImageJ. Error bars indicate standard deviation from measurement of at least 5 eyes for each genotype. Student's t-test: (***) p<0.001. (C) Histogram showing the level of inr mRNA measured by quantitative RT-PCR in control and Df(3R)Exel6186/+ flies. (D) Control deletion Df(3R)ED6076 does not sensitize to FOXO overexpression. Adult eyes expressing GMR-Gal4 alone or with UAS-FOXO. +/+ indicates 2 intact copies of the inr locus. Df(3R)ED6076/+ indicates the flies with one copy of the deletion, which is otherwise similar to Df(3R)Exel6186, but does not affect inr. Histograms showing quantification of eye size of the indicated genotypes are shown at right. Error bars indicate standard deviation from measurement of at least 5 eyes for each genotype. See inr function.Genetic tests of reduced (TIF)Click here for additional data file.Figure S8Df(3R)BSC678/+ larvae. Error bars represent standard deviation from 3 independent experiments. Lower panel: Histogram showing trehalose levels in hemolymph from wandering 3rd instar control (+/+) and Df(3R)BSC678/+ larvae.No change in levels of stored glycogen and triglycerides or circulating trehalose. Histograms showing glycogen (left) and triglyceride (right) levels normalized to total protein in control (+/+) and (TIF)Click here for additional data file.Figure S9rd instar larvae expressed UAS-inr under Tubulin-Gal4 control. Error bars represent standard deviation from at least 3 independent experiments. Student's t-test: (*) p<0.05.InR overexpression modestly, but significantly, decreased levels of circulating glucose. Wandering 3(TIF)Click here for additional data file.Figure S10inr and EGFR target sty mRNAs at various stages of Drosophila development. RNA levels were determined using RNA-seq data from modENCODE (www.modencode.org) by cufflinks (cufflinks.cbcb.umd.edu). Spearman's correlation coefficient rho = 0.71 (P<0.001). E# indicates hours of embryonic development. L# indicates larval stage. P# indicates days of pupal development. ad5d indicates 5 day adult.Correlation between the levels of (TIF)Click here for additional data file.Figure S11Acute pharmacological inhibition of MAPK/ERK has no impact on insulin activity. Immunoblots to visualize the level of AKT and ERK phosphorylation in S2 cells treated with the MEK inhibitor U0126 (Promega) or in control cells. Cells were treated with dsRNA to deplete PI3K, KSR or GFP as a control and after 5 days stimulated with insulin and treated with 10 µM U0126 for 10 min as indicated. Pharmacological inhibition of MEK by U0126 was effective, as visualized by anti-P-ERK. Anti-Kinesin was used as loading control. Samples were run on the same gel, but intervening lanes have been removed as indicated.(TIF)Click here for additional data file.