When translating mRNAs are cleaved in protein-coding regions, 5′ fragments of mRNAs are detached from stop codons (i.e., nonstop mRNAs) and protected from 3′-5′ exonucleases by ribosomes stalled at ...the 3′ termini. It has been shown in yeast that the nonstop mRNA decay (NSD) machinery triggers nonstop mRNA degradation by removing stalled ribosomes in the artificial reporter mRNAs. However, it is not known well whether NSD is involved in the degradation of endogenous nonstop mRNAs in higher eukaryotes. In this work, we addressed the question of whether 5′-nonstop-mRNA fragments generated by siRNA cleavage or nonsense-mediated-mRNA decay (NMD) are degraded by the NSD pathway in Drosophila melanogaster cells by knocking down three NSD components, Pelota (a yeast Dom34 homolog), Hbs1 and ABCE1 (a ribosome-recycling factor). We found that double, but not single, knockdown of any two of these three factors efficiently stabilized nonstop reporter mRNAs and triple knockdown of Pelota, Hbs1 and ABCE1 further stabilized nonstop mRNAs in highly ribosome-associated state. These findings demonstrated that Pelota, Hbs1 and ABCE1 are crucial for NSD in Drosophila cells as in yeast for rescuing stalled ribosomes and degrading nonstop mRNAs. To our knowledge, this is the first comprehensive report to show the involvement of the NSD machinery in the clearance of mRNA 5′-fragments produced by RNAi and NMD in eukaryotes.
•Pelota, Hbs1 and ABCE1 are crucially involved in NSD in Drosophila cells.•Doubly knockdown of Pelota/Hbs1/ABCE1 depletes NSD in Drosophila cells.•NSD pathway degrades mRNA 5′-fragments produced by RNAi and NMD.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Aptamers are short single-stranded nucleic acid molecules that are selected in vitro from a large random sequence library based on their high and specific affinity to a target molecule by a process ...known as SELEX. Cell-SELEX that employs whole living cells overexpressing the defined cell surface proteins (for selection) and appropriate mock cells (for counter-selection) has been widely used as a valid and feasible method for generating aptamers against specific cell surface proteins. However, the endogenous expression of target proteins in mock cells or the heterogeneity of surface proteins between selection and counter-selection cells often impeded the isolation of proper aptamers against target proteins. To solve this problem, we developed “Isogenic cell-SELEX” (Icell SELEX in short) method, in which isogenic cell lines were manipulated for counter-selection by microRNA-mediated silencing and for selection by overexpression of target proteins. As a model experiment, we targeted integrin alpha V (ITGAV), which is a major transmembrane receptor expressed in almost all the cells, and established ITGAV-overexpressed and -downregulated HEK293 cells for selection and counter-selection, respectively. By taking advantage of a hundred-fold difference in the expression level of ITGAV between these two isogenic cell lines, we easily isolated several anti-ITGAV aptamers, whose binding to the cell-surface ITGAV was confirmed by flow cytometry with the dissociation constant of 300–400 nM range. We assume that Icell-SELEX could be applicable to a wide range of cell-surface proteins including various transmembrane proteins of biological and pharmacological significance.
•An efficient cell-SELEX strategy is developed and named Icell-SELEX.•Selection and counter-selection cells are manipulated by gene engineering.•RNA aptamers to integrin alpha V on the cell surface were isolated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced ...from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a Drosophila ovarian somatic cell line where Piwi, but not Aub or AGO3, is expressed; thus, only the primary piRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster, traffic jam (tj), a large Maf gene, was determined as a new piRNA cluster. piRNAs arising from tj correspond to the untranslated regions of tj messenger RNA and are sense-oriented. piRNA loading on to Piwi may occur in the cytoplasm. zucchini, a gene encoding a putative cytoplasmic nuclease, is required for tj-derived piRNA production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal somatic cells, tj gives rise simultaneously to two different molecules: the TJ protein, which activates Piwi expression, and piRNAs, which define the Piwi targets for silencing.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4
+
T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. The ...two most structurally related IL-17 family members, IL-17A and IL-17F, form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) complexes. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. In this study, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. Aptamers are short single-stranded nucleic acid sequences that are selected
in vitro based on their high affinity to a target molecule. One selected aptamer against human IL-17A/F, AptAF42, was isolated by repeated cycles of selection and counterselection against heterodimeric and homodimeric complexes, respectively. Thus, AptAF42 bound IL-17A/F but not IL-17A/A or IL-17F/F. The optimized derivative, AptAF42dope1, blocked the binding of IL-17A/F, but not of IL-17A/A or IL-17F/F, to the IL-17 receptor in the surface plasmon resonance assay
in vitro. Consistently, AptAF42dope1 blocked cytokine GRO-α production induced by IL-17A/F, but not by IL-17A/A or IL-17F/F, in human cells. An RNA footprinting assay using ribonucleases against AptAF42dope1 in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1 is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.
► Successful isolation of antagonistic aptamer that specifically blocks a heterodimeric form of human interleukin-17A/F.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are the first-line treatment for exudative age-related macular degeneration (nAMD). Due to the limitations of these ...standard therapies, targeting alternative mechanisms of action may be helpful for treatment of this very common disease. Here, we investigated an anti-fibroblast growth factor-2 (FGF2) aptamer, umedaptanib pegol, a next generation therapeutic for the treatment of nAMD.
Three phase 2 studies were designed. First, a multicentre, randomized, double-masked TOFU study assessed the efficacy of intravitreal injections of umedaptanib pegol monotherapy or in combination with aflibercept, compared to aflibercept monotherapy in 86 subjects with anti-VEGF pretreated nAMD. Second, 22 subjects who had exited the TOFU study received 4 monthly intravitreal injections of umedaptanib pegol (extension, RAMEN study). Third, as an investigator-sponsored trial (TEMPURA study), a single-center, open-label, 4-month study was designed to evaluate the safety and treatment efficacy of umedaptanib pegol in five naïve nAMD patients who had not received any prior anti-VEGF treatment.
The TOFU study demonstrated that umedaptanib pegol alone or in combination with aflibercept did not improve best-corrected visual acuity (BCVA) and central subfield thickness (CST) over aflibercept alone. However, the change in BCVA and CST at primary endpoint was marginal in all the three treatment groups, suggesting that umedaptanib pegol is effective to prevent the disease progression. The RAMEN study confirmed the cessation of disease progression. In the TEMPURA study, naïve nAMD patients showed improvement and no further macular degeneration, with striking improvement of visual acuity and central subfield thickness in some of the patients.
These results demonstrate, for the first time, clinical proof of concept for aptamer based anti-FGF2 therapy of nAMD.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
AML1-MTG8 is a chimeric transcription factor produced by
t(8;21) chromosome translocation and causes AML. AML1-MTG8 acts as a dominant negative effector on normal AML1 protein, a key transcriptional ...regulator of hematopoietic differentiation, but its precise mechanism is not known. To analyze the function of AML1-MTG8 in leukemic cells and to explore the possibility of AML1-MTG8-targeted therapy, we designed nine small interfering RNAs (siRNAs) targeting a 25-nucleotide region spanning the fusion point of
AML1 and
MTG8. Two different siRNAs (AM2 and AM4) significantly reduced
AML1–MTG8 expression from a transfected reporter plasmid at both the mRNA and protein levels. Both siRNAs did not reduce
AML1b expression, but AM2 siRNA showed slightly reducing activity against
MTG8b mRNA that is 86% homologous to the corresponded region of
AML1–MTG8 mRNA. Moreover, using a cationic lipid reagent, the siRNAs were efficiently introduced into leukemia cell lines with
t(8;21), SKNO-1 (30–40%) and Kasumi-1 (60–70%) cells, and reduced specifically the endogenous
AML1–MTG8 expression. The siRNAs reduced neither the wild type
AML1 in Kasumi-1 cells nor wild type
MTG8b in human erythroblastic leukemia (HEL) cells. These results indicated that the two siRNAs are highly specific for the fusion mRNA. The knockdown of AML1-MTG8 in Kasumi-1 cells resulted in the activation of
p14
ARF
promoter activity and increased the expression of integrin αIIb, whose expression is related to megakaryocytic differentiation. However, the knockdown of AML1-MTG8 in Kasumi-1 cells did not inhibit the cell growth, suggesting that the siRNA-mediated knockdown of AML1-MTG8 is useful for the functional analysis of the gene, but it alone might not be sufficient for gene therapy of the leukemia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The aim of our study was to examine compliance with a daily dose of 5 mg alendronate (ALN) and 2.5 mg risedronate (RDN) in actual practice, and to determine the causes of noncompliance through a ...questionnaire. In addition, we studied the quality of life (QOL) of patients through another disease-related questionnaire. The overall compliance rate remained at approximately 40% one year after the initial dose. The rates did not differ significantly between the ALN group (783 patients) and the RDN group (491 patients). The compliances in the female group and the rheumatism group were better than in the male group and the nonrheumatism group. From the questionnaire, 36% of noncompliant patients showed adverse effects (AEs), and the other noncompliant patients stopped the medication in spite of having no AEs. A logistic regression analysis of factors that might have affected long-term compliance included AEs, an understanding of the disease, the method of ingestion, visiting medical facilities, the shape of the tablet, the cost of the drug, and the explanation of the doctor or pharmacist. This analysis showed that noncompliance occurred mainly due to AEs, the inconvenience of visiting a medical facility, unusual methods of ingestion, and a poor understanding of the disease. According to the results of the questionnaire for QOL assessment, the patients who continued the medication for more than 1 year had improved scores for pain in both the ALN and RDN groups. Osteoporotic treatment needs long-term patient compliance. To improve compliance, it is very important that doctors and pharmacists ensure that patients understand the purpose of this therapy.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
