GFP siRNA Research Articles

A GFP splicing reporter in a coilin mutant background reveals links between alternative splicing, siRNAs, and coilin function in Arabidopsis thaliana.

Coilin is a scaffold protein essential for the structure of Cajal bodies, which are nucleolar-associated, nonmembranous organelles that coordinate the assembly of nuclear ribonucleoproteins (RNPs) including spliceosomal snRNPs. To study coilin function in plants, we conducted a genetic suppressor screen using a coilin (coi1) mutant in Arabidopsis thaliana and performed an immunoprecipitation-mass spectrometry analysis on coilin protein. The coi1 mutations modify alternative splicing of a GFP reporter gene, resulting in a hyper-GFP phenotype in young coi1 seedlings relative to the intermediate wild-type level. As shown here, this hyper-GFP phenotype is extinguished in older coi1 seedlings by posttranscriptional gene silencing triggered by siRNAs derived from aberrant splice variants of GFP pre-mRNA. In the coi1 suppressor screen, we identified suppressor mutations in WRAP53, a putative coilin-interacting protein; SMU2, a predicted splicing factor; and ZCH1, an incompletely characterized zinc finger protein. These suppressor mutations return the hyper-GFP fluorescence of young coi1 seedlings to the intermediate wild-type level. Additionally, coi1 zch1 mutants display more extensive GFP silencing and elevated levels of GFP siRNAs, suggesting the involvement of wild-type ZCH1 in siRNA biogenesis or stability. The immunoprecipitation-mass spectrometry analysis reinforced the roles of coilin in pre-mRNA splicing, nucleolar chromatin structure, and rRNA processing. The participation of coilin in these processes, at least some of which incorporate small RNAs, supports the hypothesis that coilin provides a chaperone for small RNA trafficking. Our study demonstrates the usefulness of the GFP splicing reporter for investigating alternative splicing, ribosome biogenesis, and siRNA-mediated silencing in the context of coilin function.

Identification of RNA silencing suppressor encoded by wheat blue dwarf (WBD) phytoplasma.

Plants possess an innate immune system for defence against pathogens. In turn, pathogens have various strategies to overcome complex plant defences. Among diverse pathogens, phytoplasmas are associated with serious diseases in a range of species. RNA silencing serves as an efficient defence system against pathogens in eukaryotes but can be interrupted by RNA silencing suppressors (RSSs) encoded by pathogens. Currently, many RSSs have been identified in viruses, bacteria, oomycetes and fungi. Phytoplasmas are pathogens in several hundred plant species. In this research, 37 candidate effectors of wheat blue dwarf (WBD) phytoplasma were screened for presence of RSS. Agro-infiltration assay, yeast expression system, floral-dip method for constructing transgenic A. thaliana, Western blotting and RT-qPCR were used for identification of RNA silencing suppressors. SWP16 encoded by WBD phytoplasma was found to be a secretory protein that inhibited accumulation of GFP siRNA and led to the accumulation of GPF mRNA in systemic N. benthamiana 16c. Furthermore, in A. thaliana SWP16 inhibited production of miRNAs, which are components of RNA silencing. SWP16 also promoted infection of potato virus X. We conclude that SWP16 encoded by WBD phytoplasma was an RSS, suppressing systemic RNA silencing. This is the first evidence that a phytoplasma encodes an RSS and provides a theoretical basis for research on the interaction mechanisms between pathogens and plants.

Mutational analysis of Arabidopsis thaliana ABCE2 identifies important motifs for its RNA silencing suppressor function.

ATP‐binding cassette sub‐family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function.Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light.Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N‐terminal iron–sulphur cluster domain of AtABCE2 is crucial for its suppressor function.Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Co-delivery of pretubulysin and siEG5 to EGFR overexpressing carcinoma cells

Small interfering RNA (siRNA) represents a new class of therapeutic agents. Its successful intracellular delivery is a major challenge. Lipo-oligomeric carriers can complex siRNA into lipopolyplexes and thus mediate its cellular uptake. In this study, siRNA against the kinesin related mRNA EG5 gene (siEG5) and the microtubule inhibitor pretubulysin (PT) were co-formulated into polyplexes using azide-containing lipo-oligomer 1198. Nanoparticles were further modified by click reaction using shielding agent DBCO-PEG or EGFR targeting peptide GE11 (DBCO-PEG-GE11). Polyplexes displayed efficient payload incorporation and homogenous particle sizes of 200 nm. The biological effects of the unmodified and surface-functionalized polyplexes were investigated. The successful GE11-mediated intracellular delivery of siRNA into the EGFR overexpressing KB and Huh7 cell lines facilitated potent silencing of an EGFP-luciferase reporter gene by GFP siRNA. Specific downregulation of EG5 mRNA by siEG5 resulted in the expected antitumoral activity. The combination formulation 1198 siEG5 + PT provided superior antitumoral activity over free PT and 1198 siEG5.

Cell cycle-regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution.

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.

In vivo siRNA distribution and pharmacokinetics assessed by nuclear imaging are modulated according to radiolabelling site

IntroductionRNA interference is efficient in in vitro studies, and appears as a therapeutic tool of major clinical interest. Nevertheless, the clinical utilisation of siRNAs is restrained by the poor availability of biodistribution data on this new class of pharmaceutics. This study aimed at defining the biodistribution and pharmacokinetics properties of an siRNA directed to the Casein Kinase-2 beta (CK2β) subunit, a potential target in cancer therapy. MethodsFour CK2β siRNAs were chemically modified on each extremity of sense or anti-sense strand and radioiodinated. The biodistribution of each entity was analysed in glioblastoma-bearing mice using nuclear imaging and compared to a control GFP siRNA. ResultsThe labelling process was associated with preservation of interference activity, except when applied to the 5’ antisense terminus. Radioactivity was predominantly observed in organs of the excretory system after intravenous administration: liver, kidneys and bladder. Tumor/Contralateral muscle ratio showed significant differences depending on the labelling site. Activity associated with CK2β5’s was quite constant over 2hours, while CK2β3’as activity decreased by 40% in tumor. Finally, synchrotron X-ray analysis showed that CK2β3’s is more abundant in tumor than in liver, brain or muscle, and uniformly distributed between intra- and extracellular compartments. ConclusionsIn this study, we highlighted the large influence of siRNAs radiolabelling position on their biodistribution and pharmacokinetic profiles, and proposed a systematic approach for the imaging of all siRNAs of clinical interest.

Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers.

In plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 (DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells.

Stimuli-responsive siRNA carriers for efficient gene silencing in tumors via systemic delivery.

Development of efficient carriers for small interfering RNA (siRNA) delivery and validation tools for assessing in vivo RNA interference (RNAi) efficiency is crucial to advance RNAi-based therapeutics to the clinic. Here, acid-degradable ketalized linear polyethylenimine (KL-PEI) designed for efficient, stimuli-responsive, and biocompatible siRNA delivery was used to complex with GFP-silencing siRNA (GFP siRNA) for in vivo RNAi. The in vivo gene silencing efficiency of GFP siRNA/KL-PEI polyplexes was evaluated at mRNA, protein, and histological levels using a mouse bearing a GFP-expressing tumor. Intravenously injected GFP siRNA/KL-PEI polyplexes significantly reduced GFP expression in tumors and whole blood of mice, depending on the dosage of GFP siRNA and the time course. Average GFP mRNA levels in the tumors of siRNA/KL-PEI polyplex-injected mice were also reduced. The described siRNA carriers and RNAi validation methodologies in this study may provide insightful clues for the development of RNAi-based therapeutics and preclinical trials.

Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.

The use of anti-COX2 siRNA coated onto PLGA nanoparticles loading dexamethasone in the treatment of rheumatoid arthritis

In drug delivery systems, some genes have the potential to interrupt unnecessary gene expression in specific target cells. In this study, two types of drug, glucocorticoids and siRNA, were co-delivered into conditioned cells to inhibit the expression of unnecessary genes and proteins involved in arthritis. To deliver the two factors into a human chondrocyte cell line (C28/I2), dexamethasone was first loaded into PLGA nanoparticles, and then drug-loaded PLGA nanoparticles were complexed with poly(ethyleneimine) (PEI)/siRNA. To test the co-delivery of siRNA and dexamethasone into chondrocytes, cells were transfected with green fluorescence protein siRNA (GFP siRNA) and drugs. After transfection with GFP siRNA, 70% reduction of C28/I2 cells demonstrated GFP expression, whereas MOCK carrying PLGA nanoparticles and PLGA nanoparticles without siRNA showed no differences of GFP expressions. COX-2 and iNOS productions in C28/I2 cells were examined after TNF-α pre-treatment to induce expression of arthritis-related molecules in vitro. The reduction of gene and protein expression associated with arthritis by transfection with dexamethasone-loaded and COX-2 siRNA-complexed PLGA nanoparticles was evaluated by RT-PCR, real time-qPCR, immunoblotting, immunohistochemistry, and immunofluorescence imaging.

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

Inhibition of VEGF expression in A431 and MDA-MB-231 tumour cells by cationic lipid-mediated siRNA delivery

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid (), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.

A Somatically Diversified Defense Factor, FREP3, Is a Determinant of Snail Resistance to Schistosome Infection

Schistosomiasis, a neglected tropical disease, owes its continued success to freshwater snails that support production of prolific numbers of human-infective cercariae. Encounters between schistosomes and snails do not always result in the snail becoming infected, in part because snails can mount immune responses that prevent schistosome development. Fibrinogen-related protein 3 (FREP3) has been previously associated with snail defense against digenetic trematode infection. It is a member of a large family of immune molecules with a unique structure consisting of one or two immunoglobulin superfamily domains connected to a fibrinogen domain; to date fibrinogen containing proteins with this arrangement are found only in gastropod molluscs. Furthermore, specific gastropod FREPs have been shown to undergo somatic diversification. Here we demonstrate that siRNA mediated knockdown of FREP3 results in a phenotypic loss of resistance to Schistosoma mansoni infection in 15 of 70 (21.4%) snails of the resistant BS-90 strain of Biomphalaria glabrata. In contrast, none of the 64 control BS-90 snails receiving a GFP siRNA construct and then exposed to S. mansoni became infected. Furthermore, resistance to S. mansoni was overcome in 22 of 48 snails (46%) by pre-exposure to another digenetic trematode, Echinostoma paraensei. Loss of resistance in this case was shown by microarray analysis to be associated with strong down-regulation of FREP3, and other candidate immune molecules. Although many factors are certainly involved in snail defense from trematode infection, this study identifies for the first time the involvement of a specific snail gene, FREP3, in the phenotype of resistance to the medically important parasite, S. mansoni. The results have implications for revealing the underlying mechanisms involved in dictating the range of snail strains used by S. mansoni, and, more generally, for better understanding the phenomena of host specificity and host switching. It also highlights the role of a diversified invertebrate immune molecule in defense against a human pathogen. It suggests new lines of investigation for understanding how susceptibility of snails in areas endemic for S. mansoni could be manipulated and diminished.

Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.

Production of dsRNA sequences in the host plant is not sufficient to initiate gene silencing in the colonizing oomycete pathogen Phytophthora parasitica.

Species of the oomycete genus Phytophthora are destructive pathogens, causing extensive losses in agricultural crops and natural ecosystems. A potential disease control approach is the application of RNA silencing technology which has proven to be effective in improving plant resistance against a wide range of pests including parasitic plants, nematodes, insects and fungi. In this study, we tested the potential application of RNA silencing in improving plant disease resistance against oomycete pathogens. The endogenous P. parasitica gene PnPMA1 and the reporter gene GFP were used to evaluate the potential application of host induced gene silencing (HIGS). The GFP-expressing P. parasitica efficiently colonized Arabidopsis thaliana lines stably expressing GFP dsRNA and showed no obvious decrease in GFP signal intensity. Quantitative RT-PCR analyses showed no significant reductions in the abundance of GFP and PnPMA1 transcripts in P. parasitica during colonization of A. thaliana lines stably expressing GFP and PnPMA1 dsRNAs, respectively. Neither GFP siRNAs nor PnPMA1 siRNAs produced by transgenic plants were detected in P. parasitica re-isolated from infected tissues by Northern blot analyses. Phenotypic characterization of zoospores released from infected plant roots expressing PnPMA1 dsRNA showed no motility changes compared with those from wild-type plants. Similar results were obtained by analysis of zoospores released from sporulating hyphae of P. parasitica re-isolated from PnPMA1 dsRNA-expressing plant roots. Thus, the ectopic expression of dsRNA sequences in the host plant is not sufficient to initiate silencing of homologous genes in the colonizing oomycete pathogen, and this may be due to a number of different reasons including the absence of genetic machinery required for uptake of silencing signals in particular dsRNAs which are essential for environmental RNA silencing.

Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling

Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. Although the persistent secretion of inflammatory mediators by the activated cells contributes to the pathogenesis of various neurological disorders, most activated microglia eventually undergo apoptosis through the process of activation-induced cell death (AICD). The molecular mechanism of AICD, however, has remained unclear. Here, we show that Daxx and mammalian Ste20-like kinase-1 (MST1) mediate apoptosis elicited by interferon-γ (IFN-γ) in microglia. IFN-γ upregulated the expression of Daxx, which in turn mediated the homodimerization, activation, and nuclear translocation of MST1 and apoptosis in microglial cells. Depletion of Daxx or MST1 by RNA interference also attenuated IFN-γ-induced cell death in primary rat microglia. Furthermore, the extent of IFN-γ-induced death of microglia in the brain of MST1-null mice was significantly reduced compared with that apparent in wild-type mice. Our results thus highlight new functions of Daxx and MST1 that they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-γ.

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Novel vectors for retrotransposon-induced gene silencing in Magnaporthe oryzae

Here we report a novel system to induce RNA silencing in Magnaportheoryzae using the retrotransposon MAGGY. The gene-silencing vector pSilent-MG carries a MAGGY variant with a unique cloning site in the 3′ UTR region and a geneticin-resistance cassette as a selection marker. A gene-silencing assay with a GFP gene revealed that pSilent-MG vectors induced gene silencing at a practical level. In the resulting GFP-silenced transformants, GFP siRNAs accumulated and DNA methylation increased in the GFP-expressing gene, implying that the vector could induce both transcriptional and post-transcriptional gene silencing.

Endocytic Recycling Proteins EHD1 and EHD2 Interact with Fer-1-like-5 (Fer1L5) and Mediate Myoblast Fusion

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.