Arabidopsis thaliana, RNA-free proteinaceous RNase Ps (PRORPs) seem to be enzyme(s) for pre-tRNA 5′-end processing in organelles and the nucleus and are thought to have replaced the ribonucleoprotein RNase P variant. However, the evolution and function of plant PRORPs are not fully understood. Here, we identified and characterized three PRORP-like proteins, PpPPR_63, 67, and 104, in the basal land plant, the moss Physcomitrella patens. PpPPR_63 localizes to the nucleus, while PpPPR_67 and PpPPR_104 are found in both the mitochondria and chloroplasts. The three proteins displayed pre-tRNA 5′-end processing activity in vitro. Mutants with knockout (KO) of the PpPPR_63 gene displayed growth retardation of protonemal colonies, indicating that, unlike Arabidopsis nuclear RPORPs, the moss nuclear PpPPR_63 is not essential for viability. In the KO mutant, nuclear-encoded tRNAAsp (GUC) levels were slightly decreased, whereas most nuclear-encoded tRNA levels were not altered. This indicated that most of the cytosolic mature tRNAs were produced normally without proteinaceous RNase P-like PpPPR_63. Single PpPPR_67 or 104 gene KO mutants displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. However, the levels of all other tRNAs were not altered in the KO mutants. In addition, in vitro RNase P assays showed that PpPPR_67 and PpPPR_104 efficiently cleaved chloroplast pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency. This suggests that the two proteins have overlapping function but their substrate specificity is not identical.RNase P is a ubiquitous endonuclease that removes the 5′ leader sequence from pre-tRNAs in all organisms. In  Arabidopsis thaliana, three MRPP3 homologs have been identified as RNase P enzymes; they are termed proteinaceous RNase P 1 (PRORP1), PRORP2, and PRORP3 In vitro cleavage assays using recombinant PRORP proteins have demonstrated that three PRORP proteins display RNase P activity PRORP1 is an essential gene, because prorp1 mutants are embryonic lethal PRORP2 or PRORP3 knockout mutant lines show wild-type phenotypes, whereas the homozygous double mutation in prorp2 prorp3 results in embryonic lethality RNase P is an endonuclease that removes the 5′ leader sequence from precursor tRNAs (pre-tRNAs). This endonucleolytic cleavage is an essential step in the production of mature tRNAs in all organisms, as well as in mitochondria and chloroplasts The Arabidopsis and rice genomes do not encode RNase P RNA, whereas they do code for several homologs of RNP RNase P protein subunits found in mammals and yeasts Cyanophora paradoxa) Poryphyra purpureaNephroselmis olivacea, Ostreococcus tauriC. paradoxa, RNase P activity in an extract of the photosynthesis organelle (cyanelle) is sensitive to micrococcal nuclease O. tauri has been shown to cleave the 5′ leader of pre-tRNA in vitroUnlike in land plant organelles, an RNA subunit of bacterial-type RNase P is encoded in the organellar genomes of the Glaucophyta  that are members of the PPR protein family From these differences in the status of PRORP enzymes in algae and plants, the following questions have arisen: When were plant PRORP genes duplicated during plant evolution, which PRORP protein was targeted to the organelles and which to the nucleus, and was ribonucleoprotein RNase P enzyme replaced by PRORP in plants? To answer these questions, we focused on PRORPs of the basal land plant bryophyte Physcomitrella PPR proteins Chara have a single PRORP homolog, mosses and vascular plants have two or three PRORP homologs  knockin (KI) mosses. The four independent KI lines obtained showed normal growth , and GFPTo verify the organellar localization of PpPPR_67 and 104, their N-terminal sequences fused to GFP (N67-GFP and N104-GFP) were transiently expressed in the protonemata . GFP fluPhyscomitrella PRORP-like PPR proteins are RNase P enzymes, we performed in vitro RNase P assays with recombinant proteins composed of the respective PRORP-like protein fused with thioredoxin at the N-terminus and six histidines and a VP5 tag at the C-terminus  were altered to alanines. These two aspartates have been shown to be essential for the activity of Arabidopsis PRORP1 Asp  mutants of PpPPR_63, Δ63-1 andΔ63-15. Both KO lines were verified to be null mutants. Gene structure of theΔ63-1 line is given as PpPPR_63 transcripts was verified by reverse transcription (RT)-PCR . In addition, to generate the complementation lines of theΔ63-1, we transformed the Δ63 moss with a transgene encoding the full length of PpPPR_63 or the mutated version and generated F63 and M63, respectively (460 and D461) in the NYN domain were mutated to alanines (A460 and A461). In the complementation lines F63 and M63, the introduced transgene was overexpressed  (PpPPR_63 was responsible for the mutant phenotype.To investigate the (RT)-PCR . Therefoectively . In M63,xpressed . The KO ype (WT) , and theype (WT) . The combona fide RNase P, knockout of the PpPPR_63 gene could result in severely reduced accumulation of nuclear-encoded tRNAs. To investigate this possibility, we analyzed the accumulation levels of several nuclear-encoded tRNAs in theΔ63 mutant. For this analysis nuclear-encoded tRNAAsp (GUC) and tRNAGln (CUG) were chosen, because expression of these tRNAs has been demonstrated to significantly decrease in the knockdown mutants of nuclear PRORP2/3 plants Asp (GUC) in theΔ63 mutant and M63 complementation line was slightly reduced to 70% of that in the WT and F63. Whereas, tRNAGln (CUG) in the Δ63 mutant, M63 and F63 complementation lines accumulated to the same level as in the WT (Ser (AGA) and tRNALys (CUU) levels were not affected in the mutants. This suggests that PpPPR_63 PNase P activity may be limited to specific tRNA(s) including tRNAAsp (GUC).If nuclear PpPPR_63 could function as a n the WT . Similarin vivo function of the PpPPR_67 and 104 genes, we generated and characterized their KO mutants  showed little difference from the WT. To generate double KO mutants, we carried out four times transformation experiments, such as introducing the PpPPR_104 KO plasmid into the PpPPR_67 KO line (Δ67-1-17 line) and vice versa. However, no double KO mutants were obtained from the genotyping of 262 moss plants. This indicates that PpPPR_67 and 104 genes cannot be knocked out simultaneously. Since they may have redundant functions, single KO mutants are viable but double KO is probably lethal.To investigate the  mutants , S5, S6. mutants , and thef the WT . A peunuPpPPR_67 and 104 KO mutants, we performed northern blot analyses by using all organelle-encoded tRNA probes. The chloroplast tRNAArg (ACG) level significantly decreased inΔ104 but not inΔ67 orΔ63 (Phe (GAA) and mitochondrial tRNACys (GCA) that were used for the in vitro RNase P cleavage assay, were not altered in the WT and the three KO mutants  levels decreased in the Δ104 but not in theΔ67, we assumed that PpPPR_104 and PpPPR_67 would have distinct RNase P activity against chloroplast pre-tRNAArg (ACG). To test this possibility, three chloroplast pre-tRNAArg isoacceptors were used as substrates for in vitro RNase P cleavage assay. This assay revealed that PpPPR_67 and 104 efficiently cleaved pre-tRNAArg (CCG) and pre-tRNAArg (UCU) but they cleaved pre-tRNAArg (ACG) with different efficiency  level in the PpPPR_63 KO mutant was slightly decreased (to 70% of that in WT mosses), but most nuclear-encoded tRNA levels were not altered. This result indicates that most of the cytosolic mature tRNAs were produced normally, without proteinaceous RNase P-like PpPPR_63. This does not concur with the results observed from functional analysis of Arabidopsis PRORP2 and 3 In Arabidopsis, the double KO mutation of tonemata . This inAsp (GUC) level resulted in growth retardation and abnormal branch formation in the PpPPR_63 KO mutant. PpPPR_63 may have a versatile function rather than pre-tRNA 5′-end processing. Arabidopsis PRORP proteins are involved in the maturation of not only tRNAs but also mRNA or snoRNA It is unlikely that the slight reduction of the tRNAPpPPR_67 and PpPPR_104 but failed to obtain double KO mutants. Similarly, KO mutation of Arabidopsis PRORP1 was shown to result in embryonic lethality PpPPR_67 and 104 displayed different phenotypes of protonemal growth and chloroplast tRNAArg (ACG) accumulation. Chloroplast tRNAArg (ACG) significantly decreased in the PpPPR_104 KO mutant but not in PpPPR_67 KO mutants. The P. patens chloroplast genome codes for three tRNAArg isoacceptors in vitro RNase P cleavage assay revealed that pre-tRNAArg (ACG) was more efficiently cleaved by PpPPR_104 than by PpPPR_67, whereas pre-tRNAArg (UCU) and pre-tRNAArg (CCG) were cleaved with similar efficiency by both proteins. This is the first evidence that PRORP enzymes have substrate specificity of tRNA processing. The anticodon of P. patens tRNAArg (ACG) is modified that including inosine in chloroplasts, and tRNAArg (ICG) can recognize all four codons. Chloroplast tRNAArg (CCG) recognizing only the GGC codon is dispensable Arg (ACG) by PpPPR_104 is essential for the accumulation of mature tRNAArg (ACG). Therefore, the PpPPR_104 KO mutant may have displayed small protonemal colonies because of a great reduction in mature tRNAArg (ACG) levels.In this study, we generated single KO mutants of Unlike Arabidopsis PpPPR_63 did not result in a significant reduction in nuclear-encoded tRNAs. This raises the possibility that unidentified RNase P activity may contribute to pre-tRNA maturation in the moss nucleus  are encoded in the genome The last question is whether ribonucleoprotein RNase P enzyme was replaced by PRORP in both the organelles and the nucleus. In this study, we showed that disruption of  nucleus . HoweverPpPPR_63 mRNA, AB983707; PpPPR_67 mRNA, AB983708; PpPPR_104 mRNA, AB808584.The sequences reported in this paper have been deposited in the DDBJ/GenBank/EMBL databases with accession numbers: P. patens subsp. patens was grown at 25°C under continuous light (30 µmol photon m−2s−1), and genomic DNA and total cellular RNA were prepared from the moss protonemata as described previously The moss E. coli BL21 and purified as described previously DNAs corresponding to the proteins  were amplified by using the primers listed in Phe, mitochondrial tRNACys, and nuclear tRNAAsp were amplified by PCR from P. patens genomic DNA with oligonucleotide pairs T7-cpF5 and cpF3, T7-mtC5 and mtC3, and T7-nuDGTC5 and nuDGTC, respectively  were transcribed with T7 RNA polymerase (TaKaRa) for 1 h at 37°C, then digested with 5 U of RNase-free DNase I (TaKaRa) and 20 U of RNase Inhibitor (TaKaRa) for 15 min at 37°C. Synthesized pre-tRNAs were purified by phenol chloroform extraction.DNAs representing precursors of chloroplast tRNAectively . The 5′ Arg isoacceptors, the corresponding genes were cloned and in vitro transcribed. The obtained pre-tRNAs were dephosphorylated with Thermo Sensitive Alkaline Phosphatase (Promega) and were 5′-end radiolabeled with [γ32P] ATP and polynucleotidekinase (TaKaRa). Labeled RNAs were purified from the gel after 8% polyacrylamide gel electrophoresis (PAGE) containing 7M urea.For preparation of chloroplast pre-tRNAin vitro-synthesized pre-tRNA (2 µg) were incubated for 1 h at 25°C in a 20 µl mixture of 20 mM Tris-HCl (pH 8.0), 30 mM KCl, 4.5 mM MgCl2, 20 µg/ml bovine serum albumin, and 2 mM DTT. A half of the reaction mixture was separated on 8% polyacrylamide-7 M urea gels and stained with ethidium bromide.Cleavage assays were performed as described previously Arg isoacceptors, the 5′ end-radiolabeled pre-tRNAs (500 nM each) and the recombinant proteins (500 nM each) were incubated and aliquots were withdrawn at the indicated times. Dried PAGE gels were analyzed by Storm 820 .For cleavage assays of three chloroplast pre-tRNA 32P 5′-end-labeled oligonucleotides cp-F3 or mt-C3 (in vitro-cleaved RNAs were mixed and reverse transcribed with ReverTraAce (ToYoBo) for 1 h at 42°C, then digested with RNase A and precipitated with ethanol. The sequence ladders were obtained by using template DNA, the radiolabled primer cp-F3 or mt-C3, as described previously Theor mt-C3  and 200 PpPPR_63 gene was inserted in-frame into the GFP coding region of pGFPmutNTPII (http://moss.nibb.ac.jp). The 3′-flanking region of PpPPR_63 was inserted downstream of the nptII cassette of pGFPmutNTPII. The resultant plasmid p63-GFPKI . The KO plasmids p67KO-1 and p67KO-2  of  primers  and clon primers  was gene p67KO-2  and S5 wd p104KO  was genePpPPR_63 cDNA coding region (1785 bp) amplified by PCR using specific primers (SwaI site of the overexpression vector pOX9WZ1 PpPPR_63 (M63: D460A/D461A) was amplified from the F63 construct by using the primers 63DA-F and 63DA-R. Both constructs were linearized with NotI and introduced into the KO line Δ63.For F63, the  primers  was clonTotal cellular RNA (5 µg) was separated on 10% polyacrylamide-7 M urea gels and transferred to nylon membranes. Hybridization and washing were performed at 42°C. The gene-specific oligonucleotide probes  were labFigure S1Generation of PpPPR_63-GFP knockin (KI) mosses. (A) Schematic of insertion of p63-GFPKI into the 3′ terminal end of the PpPPR_63 coding region. Translated and untranslated regions are represented by black and white boxes, respectively. Primers P1 to P3 used for PCR, and the expected amplified DNA sizes, are shown. (B) PCR analysis using the indicated primer pairs. The predicted 4.5- and 1.8-kb fragments were amplified from the KI lines. (C) Protonemal colonies of wild-type (WT) and KI mosses grown for 2 weeks on BCDATG medium plates without antibiotics. KI-10 was used for further analyses Click here for additional data file.Figure S2Generation of PpPPR_63 knockout (KO) moss. (A) Schematic diagrams of the PpPPR_63 gene, the KO construct (p63KO4-13), and the generated knockout locus. A Geneticin G418-resistance gene (nptII) cassette was inserted into the 4th intron of PpPPR_63. Translated and untranslated regions are represented by black and white boxes, respectively. Primers P4 to P7 used for PCR and the expected amplified DNA sizes are shown. The DNA probe used in (C) is represented by grey bars. (B) PCR analysis using the indicated primer pairs. The predicted 1.9-, 2.2- and 5.5-kb fragments were amplified from the KO lines; 3.0 kb was amplified from the WT. (C) Total cellular DNAs (20 µg) from wild type (WT) and KO mosses were digested with SphI and hybridized with the PpPPR_63 probe in (A). The probe was labeled with DIG-dUTP. Predicted hybridized fragments were detected in WT and KO mosses.(EPS)Click here for additional data file.Figure S3Generation of PpPPR_63 complementation lines intoΔ63-1. (A) Schematic diagrams of PpPPR_63 loci of wild type andΔ63-1. Primers P8 and P9 and the expected fragment sizes for PCR analysis are shown. (B) The PpPPR_63 coding region (1.8 kb) amplified from the wild type cDNA was cloned into the SwaI site of the overexpression vector, pOX9WZ1, which harbors a rice actin promoter, a c-myc tag and the Zeocin resistance cassette. The mutated PpPPR_63 (M63: D460A/D461A) gene was modified from the F63 construct. (C) The predicted 1.8- and 3.0-kb fragments derived from the transgenic lines and wild type (WT), respectively, were amplified. The predicted 5.0-kb fragments were not amplified fromΔ63-1 under the PCR conditions. F63-3 and M63-8 plants were used for further analysis as F63 and M63, respectively, in 63-1 moss.(EPS)Click here for additional data file.Figure S4Generation of PpPPR_67 knockout (KO) lines. (A) Schematic diagrams of the PpPPR_67 gene (WT), the KO construct (p67KO-1), and the generated KO locus. A Geneticin G418-resistance gene (nptII) cassette was inserted into the HindIII site of the 4th exon of PpPPR_67. Primers used for PCR and the expected amplified DNA sizes are shown. (B) The predicted 5.0-, 2.1- and 1.6-kb fragments were amplified from the transgenic lines, and 3.0 kb was amplified from the WT. The KO line 67-1-17 was used in (EPS)Click here for additional data file.Figure S5Generation of PpPPR_67 knockout (KO) lines. (A) Schematic diagrams of the PpPPR_67 locus, the KO construct (p67KO-2), and the targeted KO locus. A Geneticin G418-resistance gene (nptII) cassette was inserted into the StuI site of the 5th exon of PpPPR_67. Primers and the amplified DNA sizes for PCR analysis are shown. (B) The predicted 5.0-, 2.3- and 1.5-kb fragments were amplified from the transgenic lines, and 3.0 kb was amplified from the WT. The KO line 67-2-2 was used asΔ67 in (EPS)Click here for additional data file.Figure S6Generation of PpPPR_104 knockout (KO) line. (A) Schematic diagrams of the PpPPR_104 locus, the KO construct (p104KO), and the targeted KO locus. A Geneticin G418-resistance gene (nptII) cassette was inserted into the BglII site of the 4th exon of PpPPR_104. Primers used for PCR and the expected fragment sizes are shown. (B) The predicted 5.0-, 1.8- and 1.3-kb fragments were amplified from the transgenic lineΔ104-7, and 3.0 kb was amplified from the wild type (WT). The KO line 104-7 was used asΔ104 in (EPS)Click here for additional data file.Figure S7Northern blot analysis of chloroplast tRNAs. Total cellular RNAs (5 µg) from wild type (WT), Δ67-1-17, Δ67-2-2, andΔ104-7 protonemata were separated on 8% polyacrylamide containing 7 M urea and transferred to nylon membranes. Chloroplast tRNA gene-specific oligonucleotide probes (e probes  were lab(TIF)Click here for additional data file.Figure S8Northern blot analysis of mitochondrial tRNAs. Total cellular RNAs (5 µg) were subjected to northern blot analysis as described in (TIF)Click here for additional data file.Table S1List of PRORP and PRORP-like proteins from various organisms.(XLSX)Click here for additional data file.Table S2Oligonucleotides used in this study.(XLSX)Click here for additional data file.


MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a “seed region” of moR-21 as well as a “seed match region” in the target gene 3’UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active. Ciona intestinalis as approximately 20-nt-long RNAs that are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miRs (pri-miRs) by sequencing small RNA [MicroRNA offset RNAs (moRs) were first reported in 2009 in a simple chordate ascidian mall RNA . The dismall RNA . The expmall RNA . PreviouMice were handled in accordance with US National Institutes of Health standards, and all procedures were approved by Tufts University/Tufts Medical Center Institutional Animal Care and Use Committee (IACUC). Wild type C57/Bl6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light-controlled colony room (12 h light/dark cycle) in groups of 4 with chow diet and water provided ad libitum. The mouse carotid injury model used in this study was performed as described previously . BrieflyMouse aortic smooth muscle cell (MAoSMC) harvest and culture: MAoSMC were obtained using the explants procedure as previously published . BrieflyHuman embryonic kidney 293 (HEK293), MCF-7, NIH3T3, and mouse cardiac fibroblast (MCFB) cells were cultured in DMEM medium supplemented with 10% FBS. All cells were incubated at 37°C in humidified air containing 5% CO2.Plasmid transfection was performed with Lipofectamine LTX or Lipofectamine 2000 (Life Technologies) following the manufacturer’s protocol. Transfection with miR and moR mimetics and antisense moRs (Exiqon) was performed using RNAiMAX (Life Technologies) according to the supplier’s instructions. The final concentrations of miR/moR mimetics and antisense moRs in the Western blot experiments were 20nM and 50nM, respectively.MAoSMC were transiently transfected with miR-21, moR-21, and scrambled miR control mimetics (Life technologies) at 20nM final concentration. Cells were seeded in 96-well plates at 2000 cells/well density. Cell proliferation was measured at 0, 1, 2, 3 days post seeding using Cell TiterGlo Luminescent Cell Viability assay (Promega) following the manufacturer’s protocol. Each assay was performed in quaduplicate.−ΔΔCt method. The expression levels of target genes were measured by RT-qPCR using SuperScript III First-Strand Synthesis System (Life Technologies) and SsoFastTM EvaGreen Supermix (Bio-Rad) according to manufacturer’s instructions. The primers for each target are: Cd164: forward GTGTTCTGTAATACCTCCTACC, reverse CTGCTGATGTGACAACTGAG; Fnip1: forward TCCAACCTGCTTCATTCCACTC, reverse GAACCACTCCTAGCTCCTTGAC; Mat2a: forward TTCTCATCCATTGTCGATCTC, reverse CCTCTGATAAATTGGCTTCTTC; Rassf3: forward GGCTCTGCTCAGGAAGTTTC, reverse ACTAAGGGTGTCGGTTCTGG; Mgrn1: forward CCTCTATTGATGATGTCCTG, reverse CTTACTCCTCTATACCAACAG; Trp53inp1: forward GTCACTACTTCTTCCAGCCAAG, reverse CTGAGGACTCTTCACCAATGTC; Txndc5: forward TGAGCCCACGGGTGACAAGG, reverse GCCACACCACGGAGCATAGAAC; Vps54: forward GCTCACTCGCCTGACAGATC, reverse GCAGACACCGTGAAGAGAGG; Bmf: forward ACAACTCGGAGGCTGAGAC, reverse TCTGACTGGAACACATCATCTTC; Lrig1: forward TGTTGGATACACTAGAGAGC, reverse AGTCACTACATACAGCAGAG; Stt3b: forward AGAGTTCCGAGTAGACAAAGC, reverse CCAATCTCAGCATTACGTGTTC; B2M: forward TTCTGGTGCTTGTCTCACTGA, reverse CAGTATGTTCGGCTTCCCATTC; Gapdh: forward GCCGGTGCTGAGTATGTCGT, reverse GGCGGAGATGATGACCCTTT. All the RT-qPCR experiments were done in biological replicates (n≥3). Duplicate technical replicates were done for each biological sample. Expression of each target gene was measured in duplicate and B2M or Gapdh was used as internal controls for input RNA. The relative expression of each gene was calculated using 2−ΔΔCt method. RT-qPCR was performed using a Taqman 7900HT instrument (Life Technologies).Total RNA was extracted from MAoSMC using the miRNeasy kit as described in the manufacture’s protocol (Qiagen). Concentration of the isolated RNA was quantified using a spectrophotometer. The expression levels of miR-21 and moR-21 were measured by RT-qPCR using TaqMan MicroRNA Reverse Transcription Kit and Taqman® MicroRNA Assay (Life technologies). Endogenous small nucleolar RNA 202 (snoRNA202) and U6 were used as an internal control for the amount of input RNA. Each assay was performed in duplicate. The relative expression of miR-21/moR-21 was calculated using the 2http://ycga.yale.edu/). moR quantitation was performed essentially as previously described [Total RNA including small RNAs was extracted from cultured mAoSMC using miReasy kit (Qiagen) according to the manufacturer’s instructions. Triplicate samples were prepared. Small RNA library construction and illumina HiSeq 75bp single read sequencing were performed at the Yale Center for Genome Analysis  were alP-values. Raw and processed data is available at GEO under accession number GSE75114.MAoSMC were transfected with scrambled control or moR-21 mimetics at 5nM final concentration. Triplicate samples were prepared for each treatment. Total RNA was isolated at 48hr post-transfection. Labeling and hybridization to MouseWG-6 v2.0 Expression BeadChip arrays (llumina) were performed according to the YCGA protocol. The gene expression studies were carried out using the standard protocols for MouseRef-8 v2.0 Expression BeadChip at YCGA. Briefly, the Beadstudio suite of programs were used to calculate the quantile normalized expression values for all probe sets. Bioconductor packages Lumi  and LimmGAGCTCGAGACCCCGGGGAAGTCAT-3’ (SacI site underlined), 5’- CATCTAGA CATTTAAGCAAAGACCAGAC (XbaI site underlined), with mouse genomic DNA as template. The PCR fragments were cloned into pIS0 using SacI/XbaI sites. To generate pIS0-Txndc5-mut1 and mut2, overlap extension PCR was performed as previously described [TGAATTTGGTTTATACTTTCTAA-3’, Mut1R: 5’-TTAGAAAGTATAAACCAAATTCAACCACTTGAGTTTTCTTTTCA-3’ and Mut2F: 5’-TATGAAAAGAAAACTCAAGTGACACAATTTGGTTTATACTTTCT -3’, Mut2R: 5’-AGAAAGTATAAACCAAATTGTGTCACTTGAGTTTTCTTTTCATA -3’. The resulting extended fragment was digested by BamHI and inserted between the two BamHI sites of pIS0-Txndc5. DNA sequencing was performed to confirm the mutagenesis.The luciferase reporter pIS0 was a gift from Dr. David Bartel (Addgene plasmid #12178) . To geneCTCGAGccgaTCCGACTCCTGGTACAGgcgtTCCGACGTATGGTACAGacgcTCCGACCATTGGTACAGtcgaTCCGACGTCTGGTACAGaccgTCCGACGCATGGTACAGccggTCCGACCGCTGGTACAGacgaTCCGACCTCTGGTACAGacggatcgcGGGCCCtaatatc-3’. XhoI and ApaI sites were introduced for cloning. The moR-21 sponge DNA fragment was then cloned into pCMV-d2eGFP-21 , which is a miR-21 sponge plasmid [XhoI and ApaI). The control sponge plasmid, pCMV-d2eGFP-CXCR4, was a gift from Dr. Phil Sharp (Addgene plasmid #21967) [The moR-21 sponge oligonucleotide and reverse complement strand were synthesized at IDT  as a gBlocks Gene Fragment. The sequence was as follows: 5’- actagca plasmid , using t #21967) .HEK293 were transiently co-transfected with the pIS0 luciferase reporters, a β-galactosidase reporter, and wild type or mutated moR-21 mimetics. 24hrs after transfection, a firefly luciferase assay was carried out as follows: 20μl cell lysate was added to 100μl of firefly luciferase assay buffer (Promega). The samples were placed in a luminometer  and light output was determined over a 10 second interval. β-galactosidase activity was measured with a Tropix Galacto-Light Plus kit (Applied biosystems) following the manufacturer’s instruction. Firefly luciferase activity was normalized to β-galactosidase activity.7 cell equivalents per IP), 5μL of either a normal mouse IgG  or Anti-Ago2 antibody , were used together with the Magna RIP RNA-binding protein immunoprecipitation kit, as per manufacture’s protocol (Millipore). Purified Ago2 associated RNA was subjected to qRT-PCR for detection of miR-21, moR-21 and Txndc-5.RIP was performed using HEK293 cell lysate . We detected the expression of 34 moRs coming from 32 precursors (at an expression level of >1 read per million reads (RPMR), To determine whether moR-21 plays a role in gene regulation, we performed gene profiling analysis in VSMC treated with moR-21 mimetics . As a negative control, cells were transfected with a random sequence miR mimetic. 838 transcripts were differentially expressed, including 460 that were down-regulated and 378 that were up-regulated  . To idenhttp://www.targetscan.org). We scanned mouse gene 3’UTRs for potential “seed match” sequences to nucleotides 2–8 of moR-21. 97 conserved targets with 99 conserved sites were predicted for moR-21 and 50 of these predicted targets were detected in the VSMC gene array with a signal above background noise. Among these predicted 50 targets, 24 were down-regulated in moR-21 treated cells, and 26 were unchanged in the microarray . We further compared these pathways and found that 4 pathways are shared by miR-21 and moR-21 predicted targets  in HEK293 cells and compared miR-21 and moR-21 abundance in IgG control RIP and Ago2 RIP using qRT-PCR. We found that both miR-21 and moR-21 were significantly enriched in Ago2 RIP compared with IgG RIP  . Within  IgG RIP , indicat IgG RIP .MoRs are small RNAs that were previously known to be created from the same pri-miRs as mature miRs. However, there was no prior experimental evidence that moRs had any biological functions. Our results show, for the first time, that moR-21 plays a role in post-transcriptional gene regulation and inhibits VSMC proliferation. Our findings also indicate that miR-21 and moR-21 may regulate different genes in the same pathway and may oppose each other on regulation of certain genes, such as Igf1r. MoR-21 may regulate genes through several mechanisms. For Txndc5, moR-21 may induce mRNA degradation. For Igf1r, moR-21 may play a role in translational repression (as Igf1r mRNA was unchanged by moR-21 in the VSMC microarray study). Importantly, since miRs and moRs are derived from the same precursor and are physically located next to each other, our findings also highlight the need for reinterpretation of previous miR functional studies in which the expression of miRs was manipulated through disruption of miR loci or through over-expression of complete pri-miR sequences. In these cases, expression of moRs were also likely altered and thus the observed phenotype may have resulted from changes in both miR and moR expression. Though the current findings identify moR-21 as a previously unrecognized regulator of gene expression, they also leave many important questions unanswered. Future studies are needed to confirm whether the gene regulation mechanisms described here are also utilized by other moRs. In addition, moR-mediated gene regulation may be much more complicated than described here. In the current study, we predicted moR-21 targets by searching for “seed match” sequences only in the 3’UTR. It will be of great interest to study whether moRs could base pair with other regions of mRNA, or even noncoding RNAs, and regulate their expression. Furthermore, in the current study, although Txndc-5 was verified as a direct target of moR-21 by extensive functional studies, additional detailed molecular analyses of other potential moR-21 target genes will be important, in future studies, to further explore the functional significance of moR-21. Note also, that while the current study suggests that moR-21 promotes Txndc-5 mRNA degradation through interaction with Ago2 (in a mechanism similar to that used by miRs), future studies will be necessary to provide further details of moR-21 gene regulatory mechanisms. Our study also raises unanswered questions such as why the expression levels of moRs and miRs can be so different, and how their biogenesis and degradation are regulated. Despite these limitations and unanswered questions, taken together, the current findings reveal a new level of intricacy in the regulation of gene expression by identifying moRs as biologically active participants in the regulation of gene expression.S1 Figt-test. NS: non-significant, * P<0.05; **: P<0.01.Expression levels of moR-21 (A) and miR-21 (B) in mAoSMC, HEK293, NIH3T3, MCFB were measured by qRT-PCR and normalized to the ubiquitously expressed U6 small RNA. (C) The ratio of miR-21 expression to moR-21 expression also varied by cell type. Data are from at least 3 independent experiments. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s (TIF)Click here for additional data file.S2 Figt-test. NS: non-significant, **: P<0.01, ***: P<0.001.Representative western blots are presented. Densitometry analysis for western blots is shown at the bottom. Data are from at least 4 independent experiments and presented as fold change. Values are mean ± SEM. The significance of differences between different treatments was determined by Student’s (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableFold change is reported directly with a fold change downwards marked with a minus sign. AveExp is the log base 2 of the normalized average expression value for all 6 samples. NC1,2,3 & moR1,2,3 are the log base 2 expression values for each of the control and moR21 treated samples. Adj. p. value is the Benjamini-Hochberg adjusted p. value.(XLSX)Click here for additional data file.S3 TableThe p. value is determined as the chance of finding F or more of genes in the experimental gene set  in a network of size N, by chance, if N genes were drawn randomly from the set of all genes in their network database.(DOCX)Click here for additional data file.S4 TableThe p. value is the probability that there would be an equal or higher overlap of the experimental gene set with the functions annotation gene set by chance, as determined by the Fisher’s exact test. The range is the range of observed p. values for overlap of the experimental gene set with each separate “functions annotation” set of genes included in the larger biological function category.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


A reference preparation for simian immunodeficiency virus (SIV) RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats, including minority species, were determined. This information will inform SIV research investigations and aid evaluation and development of amplification assays for SIV RNA quantification. Macaca fascicularis), a high-titer bulk SIV positive plasma stock recovered at the peak of virus replication in vivo was developed into an SIV RNA reference panel (C1−C5), based on the SIVmac251/32H/L28 isolate  in experimental design. In cynomolgus macaques . Reverse transcription and amplification reactions were performed with the SuperScript III One-Step RT-PCR system with Platinum Taq HiFi (Invitrogen) using primer pairs designed to generate five overlapping amplicons. Primers were designated as follows: 1F: CAGTCGCTCTGCGGAGAGGCTGGC; 1R: CAAGCTTAGTACTACTGGTCTCCTCCAAAGAG; 2F: TAGCTGTCTTTTATCCAGGAAG; 2R: TCTAATTAACCTACAGAGATGTTTGG; 3F: AAAATTGAAGCAGTGGCCATTAT; 3R: TACTTATGAGCTCTCGGGAACCT; 4F: GGCATAGCCTCATAAAATATCTG; 4R: GCGAGAAAACCCAAGAACCCTAG; 5F: CAGTCACCATTATGTCTGGATTG; 5R: CAAGAAAGTGGGCGTTCCCGA.de novo assembly was performed on quality-trimmed and paired reads using Geneious version 7 software. Resultant contiguous sequences were aligned with the SIVmm32H reference sequence (D01065), and a consensus sequence was generated. All reads were mapped against the consensus to establish read depth and minority species.Bar-coded libraries of amplicons were made using Nextera XT library preparation kits (Illumina), and equimolar amounts of each sample were sequenced in 250-bp paired-end MiSeq sequencing reactions. After removal of primer and adaptor sequences, gag, pol, vif, vpr, tat, rev, vpx, env, and nef), as well as the U5 region of the 5′-long terminal repeat (5′-LTR) and U3 and R regions of the 3′-LTR of the SIV genome. BLAST analysis  derived from a viral RNA template, which will augment the development and evaluation of SIV RNA amplification assays applied in SIV vaccine and pathogenesis studies.KU892415.The complete genome sequence of the C1 SIVmac251/32H/L28 reference used in the reference panel reported here has been deposited in GenBank under the accession number


Aims. This study aimed to investigate miR-106a expression in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients and to analyze the function of miR-106a. Materials and Methods. miR-106a expression levels in PBMCs from 40 healthy controls and 56 CHB patients were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The luciferase activity assays were used to determine whether miR-106a binds to 3′UTR of IL-8. miR-106a mimics and inhibitors were transfected into healthy PBMCs. IL-8 mRNA and protein levels were detected and determined by qRT-PCR and ELISA, respectively. Results. The qRT-PCR results suggested that the PBMC miR-106a levels were decreased in CHB patients. IL-8 was augmented in CHB patients and was inversely correlated with miR-106a levels. The luciferase activity assays indicated that IL-8 is a target of miR-106a. Exogenous expression of miR-106a could significantly repress IL-8 expression at both mRNA and protein levels in PBMCs, whereas miR-106a inhibitor had the opposite effects. Conclusions. This study suggested that miR-106a is downregulated in PBMCs of CHB patients and that miR-106a may play an important role in CHB by targeting IL-8. HBV caudiseases . Cytokindiseases . Howeverdiseases .Of interest, it has been recently shown that interleukin- (IL-) 8, an important inflammatory cytokine, might be involved in the pathogenesis of HBV infectious diseases, such as CHB. IL-8 is a CXC chemokine able to elicit granulocytes, NK cells, and T cell chemotaxis at the inflammatory site . It is aPrevious data had shown that the HBV-encoded regulatory HBX protein is able to transactivate the IL-8 promoter, thereby upregulating its expression . HowevermiRNAs are demonstrated to be involved in the pathogenesis of many diseases, such as viral infection and cancer. miR-199a-5p can facilitate HCV replication by regulating prosurvival pathways . The HCMDuring viral infection, virus associated molecular patterns were recognized by Toll-like receptors (TLRs), followed by recruitment of their distinct adaptor proteins, sequentially activating signalling cascades to induce cytokine production . miRNAs Our group took advantage of miRNA expression profile to identify differentially expressed miRNAs in PBMCs from CHB patients and healthy controls (data not shown), among which miR-106a was found to be significantly downregulated. In the present study, we verified the expression of miR-106a by real-time PCR in CHB patients and investigated its possible role. Our results provide a new concept to understand the role of miR-106a in immune responses induced by HBV infection.Blood samples were collected from 56 patients with CHB with abnormal liver function as well as 40 healthy people. Patients with CHB were selected from the clinic or hospitalization unit at the Department of Infection of the Fifth Affiliated Hospital of Sun Yat-sen University during January 2012 and May 2015. The standards of diagnosis complied with the standards for diagnoses of CHB prevention and treatment guidelines recommended by liver disease of the Chinese medical association. All patients with a history and clinical features of drug-induced liver injury, alcoholic hepatitis, and steatohepatitis as well as those treated with nucleotide or nucleotide-analog antiviral or immunomodulatory drugs were excluded from this study. Patients had no history of treatment for HBV prior to the study. This study was approved by the human ethics committee of Sun Yat-sen University. Informed consent was obtained from all subjects studied.The activities of ALT were calculated spectrophotometrically in serum, using Beckman Coulter kits by autoanalyzer . The results were expressed as units per liter (Unit/L).PBMCs were isolated from heparinized blood by density gradient centrifugation with a lymphocyte separation medium. Total PBMCs RNA was isolated using the Trizol reagents as instructed , and RNA concentrations were determined with a NanoDrop instrument . RNA quality and quantity were monitored by ethidium bromide staining and by UV absorbance.μL PCR reactions consisted of 10 μL of 2 × QuantiTect SYBR Green PCR Master Mix, 2 μL of 10 × miScript Universal Primer, 2 μL of 10×miScript Primer Assay, 1 μL of RNA, and 2 μL of RNase-free water. Mammalian U6B small nuclear RNA in the PBMCs was used to normalize the miRNA expression level. The miR-106a and U6B primers were purchased from Qiagen.The expression of miR-106a was determined by miScript SYBR Green PCR kit . The miRNA amplification was conducted on a Light Cycler (Roche Diagnostics). The 20 μg of extracted RNA for each sample was treated with RQ1 RNase-free DNase  for 1 h at 37°C and used as a template for cDNA synthesis using Reverse-Transcribe Kit  in accordance with the manufacturer's protocol. The relative IL-8 expression level was determined by using the following specific primers 5′- ACTCCAAACCTTTCCACC -3′ and 5′- AACTTCTCCACAACCCTC -3′ [μL containing 10 μL SYBR qPCR Mix . The relative expression of miR-106a and IL-8 (defined as fold change) was calculated by the 2−ΔΔCt . The real-time PCR experiments were repeated for three times.For quantification of IL-8 mRNA, 1 CCTC -3′  and normThe isolated PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin in U-bottom 24-well plates at 37°C with 5% carbon dioxide. The human embryonic kidney (HEK) 293 cells were grown in DMEM medium supplemented with 10% FCS. The hsa-miR-106a mimics, hsa-miR-106a inhibitor, and unrelated sequence positive control (control mimics) and negative control (control inhibitors) were purchased from GeneCopoeia . PBMCs were transfected using Lipofectamine 2000 reagents according to the manufacturer's instructions (Invitrogen). Then, the cells were subjected to analysis of miR-106a and IL-8 mRNA expression as above. And the protein expression levels of IL-8 were measured in supernatant by ELISA .β-gal control vector was also transfected as an internal control. Then, the cells were collected and determined by using a Dual-Luciferase Reporter Assay System (Promega).To generate luciferase reporter constructs, the 3′-UTR regions of IL-8 containing the predicted target sites of miR-106a were cloned into pMIR-REPORT miRNA expression reporter vector  by using synthesized fragments (5′- CTAGTTGTTGTGAGGACATGTGGAAGCACTTTAAGTTTTTTCATCATAA -3′ and 5′- AGCTTTATGATGAAAAAACTTAAAGTGCTTCCACATGTCCTCACAACAA -3′). The corresponding mutant plasmid was also constructed with the fragments (5′- CTAGTTGTTGTGAGGACATGTGGAACCCGCAAAAGTTTTTTCATCATA A -3′ and 5′- AGCTTTATGATGAAAAAACTTTTGCGGGTTCCACATGTCCTCACAACAA -3′). For luciferase reporter assays, pMIR-IL-8-3′-UTR (WT or MUT) plasmid was cotransfected with miR-106a mimics into the HEK293 cells as previously described , 37. The−ΔΔCt method. The differences of miR-106a and IL-8 expressions were assessed by using unpaired Student't test. For determination of correlation between different variables Spearman's correlation coefficient was used. p value less than 0.05 was considered to be statistically significant.All statistical analyses were conducted with the Statistical Product and Service Solutions (SPSS) version 16.0 . The real-time PCR results were analysed using the 2r = −0.693, p = 0.000) and TBIL level     and serum IL-8 protein level   . IL-8 mR= 0.000) . In addi= 0.000) .Using the Targetscan 6.0, we found that IL-8 was one of the possible target genes for miR-106a. To demonstrate it, a luciferase assay was employed for detailed analysis. HEK293 cells were cotransfected with the IL-8 3′-UTR construct (WT) or its mutant (MUT) and miR-106a mimics or miR-106a inhibitor, followed by a measurement with the luciferase reporter assay . As exper = −0.700, p = 0.000)  .α therapy in CHB patients [γ through targeting IFN-γR1 and inhibiting its expression [miRNAs play an important role in HBV infection and HBV-related disease, such as HCC, and changes in miRNA expression patterns are important indicators of these diseases. PBMC miRNAs are also suggested to be potential biomarkers. Results from recent research have shown that the miRNA expressions of PBMC, such as hsa-miRPlus-E1063, hsa-miRPlus-E1236, ebv-miR-BHRF1-2, hsa-miRPlus-A1098, hsa-miR-1267, and hsa-miRPlus-E1163, were associated with the severity of HBV-induced liver disease and therapeutic outcome of IFN-patients . Xing etpression . Our groα. Moreover, it is strictly correlated with malignant features of tumors, a frequent event in chronic HBV. By using computational methods, we predicted that miR-106a is highly likely to interact with the 3′UTR of IL-8. To confirm whether IL-8 is a direct target of miR-106a, we subcloned IL-8 3′UTR containing the target sequences into a luciferase reporter vector, and the corresponding pMIR-3′-UTR-MUT was also constructed. The vectors were then cotransfected into the HEK293 cells with miR-106a mimics or inhibitor. It was found that miR-106a binds to 3′UTR of IL-8. Next, we transfected mimics and inhibitors of miR-106a into healthy PBMCs. Our results showed that exogenous expression of miR-106a could significantly repress IL-8 expression at both the mRNA and protein levels, whereas miR-106a inhibitor had the opposite effects. Further statistical analysis showed that miR-106a is negatively related to IL-8 expression in PBMCs from CHB patients. These data revealed that miR-106a may be an effective strategy for regulating the expression of IL-8.HBV is a typically noncytopathic virus that does not directly damage infected cells. The pathogenesis is largely mediated by immune responses following HBV infection , 5. IL-8In summary, our study firstly provides the evidence that miR-106a is downregulated in PBMCs of CHB patients, and miR-106a expression levels were negatively associated with the severity of HBV-induced liver disease. In addition, our data demonstrated that miR-106a downregulates IL-8 expression by targeting its 3′UTR. We believe that our study may help to develop a better understanding of clinical characterization and pathogenesis of CHB.


Background: G0S2 acts as an endogenous inhibitor of ATGL.Results: We designed a short peptide that is active in the nanomolar range. The G0S2-derived peptide inhibits ATGL activity in a noncompetitive manner.Conclusion: G0S2-derived peptides act as potent and specific inhibitors of ATGL.Significance: G0S2-derived peptides provide a novel tool to modulate ATGL activity. 0/G1 switch gene 2 (G0S2) is a small basic protein that functions as an endogenous inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in intracellular lipolysis. In this study, we identified a short sequence covering residues Lys-20 to Ala-52 in G0S2 that is still fully capable of inhibiting mouse and human ATGL. We found that a synthetic peptide corresponding to this region inhibits ATGL in a noncompetitive manner in the nanomolar range. This peptide is highly selective for ATGL and does not inhibit other lipases, including hormone-sensitive lipase, monoacylglycerol lipase, lipoprotein lipase, and patatin domain-containing phospholipases 6 and 7. Because increased lipolysis is linked to the development of metabolic disorders, the inhibition of ATGL by G0S2-derived peptides may represent a novel therapeutic tool to modulate lipolysis.The protein G Most organisms store excess energy as neutral and inert triacylglycerols (TGs)1–Atgl knock-out mice  hydrolyzes TGs into diacylglycerols and FAs and is required for efficient intracellular lipolysis . Global out mice . Accordiout mice . Howevertrations , 10. Consitivity . Recentlfat mass .0/G1 switch gene 2 (G0S2) was discovered in 2010 and confirmed using human and murine proteins -oleoylglycerol, oleoyl-CoA, and free glycerol detection reagents were purchased from Sigma. 1-Oleoyl-2-hydroxy-sn-glycero-3-phosphocholine was purchased from Avanti Polar Lipids Inc., Alabaster, AL, and the NEFA kit was from WAKO Diagnostics, Neuss, Germany. Hi76-0079 obtained from Novo Nordisk, Denmark, Atglistatin was a generous gift from R. Breinbauer . The protein assay kit was obtained from Bio-Rad; Thermo Scientific, Rockford, IL was the source for the Pierce® BCA protein assay kit. The synthetic peptides were synthesized by Peptide Specialty Laboratories GmbH, Heidelberg, Germany.triolein substrate together with 50 μg of cell extract of Strep-mATGL (or COS-7-mATGL) and 2.5 μg of purified mCGI-58. As a control, lysates of the His6-Smt tag were incubated under the same conditions. To determine the enzyme kinetics and the inhibitory mechanism of the hGW2052 peptide, the assay was performed with minor modifications. TG substrate was prepared with 1.67 mm triolein, 10 μCi/ml triolein (PerkinElmer Life Sciences), and 190 μm phosphatidylcholine/phosphatidylinositol (3:1) (Sigma) and diluted to the respective concentrations after sonication. Assays with CGI-58 co-activated ATGL were performed on a smaller scale. Briefly, cell lysates of Strep-mATGL  were mixed with 2.5 μg of purified mCGI-58 and 1% of the synthetic peptide hGW2052, control peptide, or DMSO in a total volume of 25 μl of sucrose solution (see above) and were incubated with 25 μl of [3H]triolein substrate. Lysates using LacZ-expressing cells were used as controls. The reaction was terminated by adding 650 μl of methanol/chloroform/heptane (10:9:7) and 200 μl of 100 mm potassium carbonate buffer, pH 10.5 (adjusted with boric acid). The radioactivity in 200 μl of the upper phase  was determined by liquid scintillation counting. Nonlinear regression analysis for inhibitor kinetics was carried out using GraphPad Prism 5 . TG hydrolase activity of murine HSL (expressed in COS-7 cells) and purified bovine lipoprotein lipase (Sigma) was determined under the same conditions.TG hydrolase activity assay was performed as described . To screrac-1-(3)-oleoylglycerol (Sigma) as substrate for 20 min at 37 °C. After incubation, the reaction was terminated by adding 100 μl of chloroform. After vigorous mixing using a vortex and centrifugation at 10,000 × g for 5 min, the glycerol release (into the upper aqueous phase) was determined using a commercial kit  according to the manufacturer's protocol.Monoacylglycerol hydrolase activity of murine MGL was determined as described . In briem substrate 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine  in a water bath at 37 °C for 20 min. Each reaction was terminated by heat inactivation at 75 °C for 10 min. The released amounts of fatty acids were determined using a commercial assay kit (HR Series NEFA-HR(2), WAKO Diagnostics) according to the manufacturer's protocol.Lysophospholipase activity of PNPLA6 and PNPLA7 was determined as described previously . In brieProtein concentrations of cell extracts were determined using the protein assay Kit according to the manufacturer's instructions (Bio-Rad) using BSA as standard. The concentrations of purified proteins were obtained by the absorbance at 280 nm using the NanoDrop® ND-1000 spectrophotometer . Alternatively, protein concentrations were determined using the Pierce® BCA protein assay kit and BSA as standard according to the manufacturer's protocol (Thermo Scientific).t test (two-tailed). Group differences were considered statistically significant for p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).All measurements were performed in triplicates. Data are presented as means ± S.D. Statistical significance was determined by the Student's unpaired A). As ATGL exhibits very low basal activity, we performed activity assays in the presence of CGI-58 to fully stimulate ATGL. Our results show that C-terminal truncations of G0S2 up to residue Val-46 completely inhibit CGI-58 activated ATGL activity  in COS-7 lysates (B). Inhibition was significantly reduced when hG0S2 was truncated at residue Leu-40 (B). These results were reproduced using proteins  expressed in E. coli (C), suggesting that other mammalian proteins are not required for G0S2-mediated inhibition. It also demonstrates that mouse ATGL is also inhibited by human G0S2. Next, we investigated the amino acid residues between Leu-40 and Val-46 and their necessity for ATGL inhibition by G0S2 in more detail. We show that the G0S2 variant Met-1–Met-43 (hG0S2_1–43) harbors the longest C-terminal deletion that still exerts the full inhibitory effect on ATGL (C). Screening various N-terminal deletions revealed that truncations of hG0S2 until residue Tyr-27 (hG0S2_27–103) fully inhibit ATGL activity (D). Further truncation of the N terminus resulted in significant loss of the inhibitory effect. Truncation of G0S2 after residue Gly-30 resulted in complete loss of the capacity to inhibit ATGL. Next, we tested the effect of a combination of N- and C-terminal truncations of hG0S2. As expected, hG0S2 Leu-10–Ala-52 and Lys-20–Ala-52 fully retained their activity to inhibit ATGL, although hG0S2 Gly-30–Ala-52 had lost all inhibitory activity. Interestingly, neither hG0S2 Tyr-27–Ala-52 nor hG0S2 Lys-20–Met-43 exhibited full inhibitory capacity (E), as extrapolation of the findings presented above would have suggested (C and D).To identify the minimal sequence within G0S2 that is strictly required to exert an inhibitory effect on ATGL activity, we tested different C- and N-terminally shortened versions of human G0S2 (hG0S2) A. As ATG lysates B. Inhibie Leu-40 B. These  E. coli C, sugges on ATGL C. Screenactivity D. Furthecapacity E, as extuggested , C and DA). A Trp residue was introduced at the N terminus of this peptide to facilitate concentration determination by UV spectroscopy. The resulting peptide was termed hGW2052. In agreement with the overexpressed G0S2 variant hG0S2_20-52 . This indicates that the synthesized peptide adopts a tertiary structure compatible for protein-protein interaction with ATGL. A control peptide was equally treated and did not show an inhibitory effect on ATGL (A and B). Next, we tested whether hGW2052 inhibits ATGL also in the absence of CGI-58. Indeed, we found that hGW2052 inhibits ATGL independently of the presence of CGI-58 (C). The dose-response experiments of basal ATGL- and CGI-58-stimulated ATGL activity using increasing amounts of hGW2052 revealed half-maximal inhibitory concentrations (IC50) of 10 and 18 nm, respectively (D and E). In agreement with these results, dose-response experiments using overexpressed, full-length hG0S2 revealed an IC50 of 19 nm (F).In our next experiments, we investigated whether synthetic peptides with sequence stretches corresponding to G0S2 are efficient in inhibiting ATGL, and we characterized the peptide-mediated inhibition kinetic of ATGL. Therefore, we designed a synthetic peptide identical to the human G0S2 sequence from Lys-20 to Ala-52 A. A Trp S2_20-52 , the synectively , A and B on ATGL , A and Bf CGI-58 C. The doectively , D and Eof 19 nm F.A) (m triolein substrate was observed in saturation kinetic experiments in the absence of an inhibitor (B). Within that substrate concentration range, we also observed a linear increase in enzyme velocity in the presence of different inhibitor concentrations (C and D). Dixon-Plot analysis (v against inhibitor concentration at each concentration of triolein substrate) resulted in a set of intersecting lines on the x axis compatible with a noncompetitive inhibition mechanism (E). Using this method, we determined an apparent iK of 28 nm for hGW2052. Nonlinear regression analysis (Graph Pad Prism) using a noncompetitive inhibition model was also used to extract kinetic parameters and resulted in an apparent iK of 25 ± 1 nm, which is in excellent agreement with the result from the Dixon-Plot method .To investigate the mode of ATGL inhibition by hGW2052, we performed enzyme kinetics. In agreement with previously published data, time course experiments showed a linear increase in FA release of ATGL-containing lysates incubated with an artificial TG substrate for up to 60 min A 13). F. FA)  and Hi 76-0079 (representing an HSL inhibitor) reduced TG hydrolase activity of WAT by 89 and 88%, respectively (A). Neither hGW2052 nor Atglistatin inhibited WAT TG hydrolase activity derived from Atgl-KO mice (A). These results indicate that hGW2052 and Atglistatin specifically inhibit ATGL but not HSL activity.Next, we investigated the specificity of the peptide hGW2052. ATGL and HSL together account for more than 95% of intracellular TG hydrolase activity in white adipose tissue (WAT) . For botectively A. Neithe-KO mice A. These B and C, hGW2052 did not exert any inhibitory effect on the lysophospholipase activities of PLNPLA6 or PNPLA7, up to a concentration of 1.4 μm. As expected, the control peptide also did not affect the lysophospholipase activity of PNPLA6 and PNPLA7 (B and C). Thus, we conclude that hGW2052 does not act as general inhibitor for members of the PNPLA protein family. We also tested for a potential effect of the inhibitory peptide hGW2052 on other extracellular (lipoprotein lipase) and intracellular (MGL and HSL) lipases. No inhibitory effect on any of these lipases was observed upon addition of up to 1.4 μm hGW2052 or control peptide, respectively (D–F). Together, these experiments demonstrate that the peptide hGW2052 is not a general lipase inhibitor and, among the different lipases tested, rather acts specifically on ATGL.As ATGL is a member of the patatin-like phospholipase domain containing protein (PNPLA) family , we furtd PNPLA7 , B and Cectively , D–F. ToAbnormally high concentrations of circulating FAs have deleterious effects on the organism, including ectopic lipid accumulation, insulin resistance, inflammation, cellular dysfunction, and cell death , 2. ATGLe.g. variants Lys-20–Met-43 or Tyr-27–Ala-52) did not result in G0S2 variants that were able to inhibit ATGL efficiently. We hypothesize that a core peptide Tyr-27–Met-43 is mainly involved in ATGL inhibition, yet a few additional residues are required to mediate full function of the inhibitory peptide.G0S2 has been identified to act as a physiological inhibitor of ATGL. However, very little is known about the molecular basis and the mode of ATGL inhibition by G0S2. Therefore, these questions were addressed in this study. We also identified minimal sequences of G0S2 that inhibit ATGL's TG hydrolase activity. Variants of G0S2 that were fully able to inhibit ATGL included extensive N- or C-terminal truncations of the wild-type protein . Interestingly, however, a combination of the identified N- and C-terminal boundaries . Consequently, physiologically relevant ATGL regulation by G0S2 is very plausible, and administration of hGW2052 at physiological concentrations can be envisaged. This high affinity of G0S2 is in contrast to the affinity of oleoyl-CoA for ATGL, which exhibits a much higher IC50 value (33 μm) . Some family members have already been characterized and exhibit TG hydrolase activity  or lysophospholipase activities (PNPLA6 and PNPLA7) . AlthougAtgl results in severe heart defects due to massive TG accumulation in cardiac muscle (–versus noncompetitive inhibition modes are additional important differences when comparing the potential impact of the peptide inhibitor hGW2052 with Atglistatin. hGW2052 inhibits ATGL in a noncompetitive manner and therefore inhibits ATGL despite high physiological substrate concentrations. Thus, hGW2052 represents a powerful research tool for tissue-selective inhibition of ATGL and has a high potential for translation into therapeutic application.Genetic disruption of c muscle , 55. Thuc muscle . This dec muscle –58, whicStructural knowledge on ATGL is very limited. It is predicted to exert lipolytic activity via a catalytic dyad that resides in the patatin domain within the N-terminal half of the lipase , 50. Pre


While A-type lamin R482W mutation is responsible for Dunnigan type familial partial lipodystrophy (FPLD2), we show that both overexpression of LMNA p.R482W in primary human preadipocytes and endogenous expression of A-type lamins p.R482W in FPLD2 patient fibroblasts, reduce A-type lamins-SREBP1 in situ interactions and upregulates a large number of SREBP1 target genes[LMNA mutant was previously shown to inhibit adipogenic differentiation, we propose that deregulation of SREBP1 by mutated A-type lamins constitutes one underlying mechanism of the physiopathology of FPLD2.SREBP1 (Sterol regulatory element binding protein 1), transcription factor that regulates hundreds of genes involved in lipid metabolism and adipocyte differentiation, is a direct partner of A-type lamins. We show that i)


Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca •Multiple biochemically distinct populations of ER-endocytic organelle contact sites•Annexin A1-S100A11 interaction tethers ER contacts with EGFR-positive endosomes•ER-to-endosome cholesterol transfer to support ILV formation requires contact sites•ER to endosome cholesterol transport depends on direct VAPA-ORP1L interaction Eden et al. identify annexin A1 as a tether for ER membrane contact sites with EGFR-containing endosomes. Under low cholesterol conditions, annexin A1-regulated contact sites are required for ER-derived cholesterol transport to endosomes. This is necessary for the formation of intraluminal vesicles within the endosomal lumen that spatially regulate EGFR signaling. The ER forms an extensive network of membrane contact sites (MCSs), microdomains of close membrane apposition (<30 nm), with a diverse range of functionally distinct organelles, providing an important means of non-vesicular communication between organelles. Although only recently described , MCSs beThe molecular composition of ER contacts with the endocytic pathway remains poorly understood, hampering functional studies. MCSs are stabilized by tethering complexes that maintain close proximity between apposing membranes. Vesicle-associated membrane protein-associated proteins (VAPs) are conserved ER membrane proteins that recruit binding partners to multiple MCSs between the ER and other organelles  by bindiWe have used electron microscopy (EM) to unequivocally identify MCSs, while also allowing the distinction between MVBs (containing discrete ILVs) and electron-dense lysosomes A. Co-incIn contrast to the effects of VAP depletion, when we depleted cells of annexin A1, MCSs between the ER and EGFR-MVBs were considerably reduced C and S1B2+-dependent heterotetramer with S100A11  . TherefoOverall these data show that the requirements for MCS and EGF-stimulated ILV formation are similar, consistent with our hypothesis that annexin A1/S100A11 tether MCSs that provide sites for PTP1B-mediated effects on ILV formation.As MVBs mature, the number of ILVs increases. Since ILVs are rich in cholesterol , we hypoIncreasing the need for sterol traffic from ER to EGFR-MVBs, by culturing cells in LPDS, increased the numbers of contacts between the two organelles B. This lHaving established a role for annexin A1-dependent transport of cholesterol from the ER to EGFR-MVBs in ILV formation, we next investigated candidate proteins that could play a role in cholesterol sensing and/or transfer at the contact. Our observation that ORP1L depletion, like LDL removal, resulted in increased MCSs specifically with EGFR-MVBs B suggest2+-dependent ligand, S100A11. In vitro studies have shown that a heterotetramer of annexin A1 and S100A11 can bridge membranes alone . Cells were transfected using Lipofectamine LTX Plus for plasmids and Lipofectamine RNAiMax (Invitrogen) for small interfering RNAs (siRNAs), according to the manufacturer's instructions.A complete description is provided in the Conventional and cryo-immunoEM were performed essentially as described  but withImmunofluorescence was essentially as previously described  with modDescribed in full in E.E., E.S.H., and A.T. conducted experiments, E.E., A.S., T.L., and C.F. designed experiments, and E.E., T.L., and C.F. wrote the paper.


Staphylococcus aureus is a Gram-positive bacterium which colonises approximately one third of the adult human population. Staphylococcal infections can be life-threatening and are frequently complicated by multi-antibiotic resistant strains including methicillin-resistant S. aureus (MRSA). Fluorodeoxyglucose ([18F]FDG) imaging has been used to identify infection sites; however, it is unable to distinguish between sterile inflammation and bacterial load. We have modified [18F]FDG by phosphorylation, producing [18F]FDG-6-P to facilitate specific uptake and accumulation by S. aureus through hexose phosphate transporters, which are not present in mammalian cell membranes. This approach leads to the specific uptake of the radiopharmaceutical into the bacteria and not the sites of sterile inflammation.Management of infection is a major clinical problem. 18F]FDG-6-P was synthesised from [18F]FDG. Yield, purity and stability were confirmed by RP-HPLC and iTLC. The specificity of [18F]FDG-6-P for the bacterial universal hexose phosphate transporter (UHPT) was confirmed with S. aureus and mammalian cell assays in vitro. Whole body biodistribution and accumulation of [18F]FDG-6-P at the sites of bioluminescent staphylococcal infection were established in a murine foreign body infection model.[In vitro validation assays demonstrated that [18F]FDG-6-P was stable and specifically transported into S. aureus but not mammalian cells. [18F]FDG-6-P was elevated at the sites of S. aureus infection in vivo compared to uninfected controls; however, the increase in signal was not significant and unexpectedly, the whole-body biodistribution of [18F]FDG-6-P was similar to that of [18F]FDG.in vitro validation, [18F]FDG-6-P did not behave as predicted in vivo. However at the site of known infection, [18F]FDG-6-P levels were elevated compared with uninfected controls, providing a higher signal-to-noise ratio. The bacterial UHPT can transport hexose phosphates other than glucose, and therefore alternative sugars may show differential biodistribution and provide a means for specific bacterial detection.Despite conclusive  This would lead to high signal-to-background ratio and enables specific diagnosis of infection, rather than sterile inflammation. Here we report on the evaluation of [18F]FDG-6-P as an imaging agent for S. aureus infection in vivo using pre-clinical PET-CT.To this end, we have exploited an alternative sugar transporter, the bacterial universal hexose phosphate transporter (UHPT) . The UHPAll animal experiments were approved by the University of Nottingham Animal Welfare and Ethical Review Board and performed in accordance with the UK Home Office Licence rules, under Project Licence 40/3821. All injected volumes were in line with ASPA guidelines.t-test  and Mann-Whitney U test . Unless otherwise stated, for in vitro data, error bars show standard error of the mean (SEM); for in vivo data, bars on graphs show data median.Statistical analysis was performed using Prism 6 . Where appropriate, analyses were performed by student's 18F]FDG-6-P was prepared from commercially available [18F]FDG  following a method previously described [18F]FDG-6-P product was determined by reverse-phase high-performance liquid chromatography (RP-HPLC) on an Agilent 1260 Series LC  connected to Flow-RAM™ sodium iodide detector . A SphereClone SAX 5 μm 250 × 4.6 mm column  was used for RP-HPLC at a flow rate of 0.8 ml min−1 with UV detection at 295 nm, using an isocratic method with 20 mmol l−1 potassium phosphate (pH 7.2) as the mobile phase. Instant thin layer chromatography (iTLC) was carried out using reverse-phase plates . Radiolabelling efficiency on iTLC strips was measured using a Bioscan radio-TLC Scanner . To ensure the stability of [18F]FDG-6-P over time, 2 MBq of [18F]FDG-6-P was incubated at 37°C for up to 3 h in 1 ml water or for 1 h in 1 ml blood (extracted from mice and heparinised). The products were analysed by HPLC (water) or iTLC (blood).FDG or 2 MBq [18F]FDG-6-P for 1 h at 37°C. Bacteria and non-adherent mammalian cells were harvested by centrifugation  and washed three times by centrifugation. All supernatants were collected for each sample in scintillation vials. After washing, the cells were transferred into scintillation vials. Adherent cell lines were washed three times by replacing the well medium. Supernatants were collected into scintillation vials. The cells were removed from the wells by trypsin treatment and placed into scintillation vials. The scintillation vials for cells and supernatants were counted by gamma counter , and the results were obtained as counts per min (cpm).For experimentation, non-adherent cell lines  and by calculating the percentage of activity in the cell containing scintillation vials compared with the total counts (cells and supernatant combined). The percentage of activity associated with the cells was normalised so that FDG  or [18F]FDG-6-P  by intraperitoneal (ip) injection. All scans were carried out 1 h later using a nanoScan PET-CT  small animal scanner with the following parameters: CT: 1 field of view (FOV), maximum FOV, full scan, 720 projections; tube 35 kVP, 170 ms exposure, 1:1 binning. PET: coincidence 1:5, scan time 20 min, packet timestamp. Scanning and subsequent reconstruction were carried out using Nucline software (Mediso). Reconstruction parameters are as follows: 3D, whole body, non-dynamic with 1:3 binning.At 24 h post bacterial inoculation, mice were injected with 10 MBq FDG-6-P and [18F]FDG measured by iTLC were 0.27 and 0.41 respectively. The stability of [18F]FDG-6-P over time was confirmed by HPLC analysis after incubation of the radiopharmaceutical in water for 3 h .The 18F]FDG-6-P to be a potential translational tool for the specific diagnosis of bacterial infections, it should not be transported into mammalian cells. Consequently, several mammalian cell lines of both human  and murine origins (HIB-1B and 3 T3-L1) were incubated with [18F]FDG or [18F]FDG-6-P, and the relative activity associated with the cells was determined . The relative amount of [18F]FDG-6-P uptake in the mammalian cell lines was comparable with that of the UHPT-deficient staphylococcal mutant, indicating there was no specific mammalian cell transport system for the phosphorylated sugar.In order for [d Figure b. For ea18F]FDG-6-P was performed (administered doses ranged between 8 and 11 MBq). The biodistribution of [18F]FDG-6-P was shown to be visually comparable with that of [18F]FDG, which had been similarly administered by ip injection into a secondary cohort of mice (administered doses ranged between 8 and 11 MBq). No uptake of [18F]FDG-6-P into host tissues was anticipated; however, as for the [18F]FDG biodistribution, [18F]FDG-6-P activity was captured from the leg muscles, heart, brown adipose tissue (BAT) and brain  and uninfected mice . The SUV values for specific organs varied per mouse; however, the trend demonstrated that infection did not greatly affect the whole-body biodistribution of either of the radiopharmaceuticals and that [18F]FDG-6-P biodistribution was not significantly different to that of [18F]FDG.Infections were confirmed by BL imaging of all mice Figure a. Whole n Figure b, as welin vivo biodistribution of [18F]FDG-6-P was highly unexpected. In order to explore whether the [18F]FDG-6-P was being dephosphorylated to produce [18F]FDG during systemic circulation, samples of mouse blood were collected and incubated with either [18F]FDG or [18F]FDG-6-P ex vivo for 1 h. The blood was analysed by iTLC to examine the peak profiles for each of the radiopharmaceuticals  and a smaller [18F]FDG peak (indicating that some [18F]FDG had not yet been transported into the cells). The blood incubated with [18F]FDG-6-P had only one defined peak, consistent with the [18F]FDG-6-P standard which had not been incubated with blood, indicating that [18F]FDG-6-P was not being dephosphorylated to [18F]FDG.The s Figure . As anti18F]FDG-6-P in vivo, the accumulation of [18F]FDG-6-P and [18F]FDG at the site of bacterial catheter foreign body infections was characterised. After image reconstruction of data captured from the nanoScan PET-CT scans  ratios were calculated for the catheter site based on the mean SUV value for each group Figure c. For ea18F]FDG imaging. During the preparation of this manuscript, Gowrishankar [18 F]-fluoromaltose [18F]-fluorodeoxysorbitol ([18F]FDS) respectively for pre-clinical imaging. [18F]FDS can be prepared from [18F]FDG, the most widely available PET imaging radiopharmaceutical and therefore [18F]FDS could potentially be used in any clinic with access to [18F]FDG. However, [18F]FDS is specific for the Gram-negative Enterobacteriaceae and so cannot be used for imaging S. aureus infections. The development of an imaging agent to detect S. aureus infections is therefore urgently required. As stated within the introduction, MRSA strains are the most widely isolated multi-antibiotic resistant bacteria found within European hospitals FDG by converting it to the corresponding phosphate analogue, [18F]FDG-6-P. The latter was confirmed as a substrate for the bacterial UHPT in in vitro accumulation assays in comparison with [18F]FDG. S. aureus RN6390 and the isogenic UHPT mutant strain accumulated [18F]FDG to the same extent, although the level of [18F]FDG-6-P in the parental cells was significantly higher. This was consistent with the elevated growth rates observed when S. aureus RN6390 was supplemented with glucose-6-phosphate instead of glucose (data not shown), suggesting that the UHPT was active and could allow even higher levels of [18F]FDG-6-P to accumulate at the site of infection compared with [18F]FDG, resulting in an elevated signal-to background ratio. Accumulation assays with [18F]FDG and [18F]FDG-6-P and various mammalian cell lines in vitro were performed to confirm that eukaryotic cells did not transport [18F]FDG-6-P. It is important to note that both murine and human-derived cell lines were used for the in vitro assays and no differences in radiopharmaceutical accumulation were observed. Therefore, it was anticipated that [18F]FDG-6-P biodistribution and accumulation in both mice and humans would be similar.Due to its accepted clinical use and widespread availability, we chose to modify [in vivo infection studies with [18F]FDG-6-P were therefore performed. A foreign body model of infection with S. aureus was used to replicate clinical situations where biofilm formation results in chronic infections that are refractory to antibiotic therapy [18F]FDG imaging [18F]FDG-6-P after clearance from the blood pool would be minimal. Mice injected with [18F]FDG were prepared identically to those injected with [18F]FDG-6-P to enable direct comparisons. All mice were imaged optically to confirm equivalent infection levels prior to [18F]FDG-6-P or [18F]FDG administration.Pre-clinical  therapy ,39,40. F imaging ,42 becau18F]FDG-6-P were observed for both infected and uninfected mice. The whole-body biodistribution of [18F]FDG and [18F]FDG-6-P was visually similar, and this was confirmed when SUVs were calculated from ROIs of the brown adipose tissue, heart, brain and right and left thighs. The first potential explanation for this finding was that the [18F]FDG-6-P was either unstable or was specifically dephosphorylated within the systemic circulation in the mouse, resulting in the release of free [18F]FDG which could then be transported into mammalian cells by GLUT transporters. However, glucose-6-phosphatase distribution in mice has previously been studied with ex vivo whole-body autoradiography [18F]FDG-6-P in the blood was also confirmed by ex vivo incubation and analysis by iTLC.Unexpectedly, very high levels of background signal from mice injected with FDG-6-P and [18F]FDG, differences between the two radiopharmaceuticals were observed at the site of the catheter implant for uninfected mice. [18F]FDG but not [18F]FDG-6-P accumulated at the ends of the catheters which are likely to be sites of sterile inflammation. It can be assumed that similar levels of inflammation were present at the ends of the catheters as all catheters were prepared and implanted in the same way. This suggests that the two radiopharmaceuticals behave differently and that [18F]FDG-6-P was not present as [18F]FDG. This raises the prospect that the observed uptake of [18F]FDG-6-P into the other organs may be a real process and so warrants further investigation to determine whether this phenomenon extends to man. Importantly, the reduced [18F]FDG-6-P activity at the catheter site of uninfected mice in this proof of concept study demonstrated that the [18F]FDG-6-P activity around the infected catheter is associated specifically with S. aureus and not inflammatory cells; however, due to the small sample size, it is not possible to determine statistical significance.Critically, despite the similarity in the whole-body biodistribution of FDG-6-P did not behave as expected within the mouse model; however, the radioactivity at the infection site was elevated more than twofold for infected mice compared with uninfected controls injected with [18F]FDG-6-P and compared with [18F]FDG. This indicated that [18F]FDG-6-P did not accumulate within the inflammatory cells and so this could conceivably provide a means to distinguish between sites of sterile inflammation and S. aureus infection, an important limitation of [18F]FDG imaging [18F] may enable the specific targeting of the UHPT, reducing background uptake and provide a more favourable biodistribution for in vivo imaging.Based on our  imaging . In addi