scholarly journals A High-Throughput Drug Screen Targeted to the 5'Untranslated Region of Alzheimer Amyloid Precursor Protein mRNA

2006 ◽  
Vol 11 (5) ◽  
pp. 469-480 ◽  
Author(s):  
Sanghamitra Bandyopadhyay ◽  
Jake Ni ◽  
Amy Ruggiero ◽  
Karen Walshe ◽  
Mark S. Rogers ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
High Throughput ◽  
Translational Control ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Internal Ribosomal Entry Site ◽  
Precursor Protein ◽  
Luciferase Reporter ◽  
Entry Site ◽  
Novel Approach

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Aβ) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Aβ production for AD.

scholarly journals A GG Nucleotide Sequence of the 3′ Untranslated Region of Amyloid Precursor Protein mRNA Plays a Key Role in the Regulation of Translation and the Binding of Proteins

2000 ◽  
Vol 20 (13) ◽  
pp. 4572-4579 ◽  
Author(s):  
E. G. Mbongolo Mbella ◽  
S. Bertrand ◽  
G. Huez ◽  
J.-N. Octave
Keyword(s):  
Amyloid Precursor Protein ◽  
Translational Control ◽  
Cho Cells ◽  
Untranslated Region ◽  
Specific Binding ◽  
Alternative Polyadenylation ◽  
Mrna Translation ◽  
Precursor Protein ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3′ untranslated region (3′UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3′UTR containing 8 nucleotides more than the short 3′UTR allows the recovery of an efficiency of translation similar to that of the long 3′UTR. Moreover, the two guanine residues located at the 3′ ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3′UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3′UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.

scholarly journals Konstruksi Plasmid Rekombinan untuk Inisiasi Translasi Enhance Green Fluorescent Protein oleh Internal Ribosomal Entry Site HIV-1

2018 ◽  
Vol 28 (2) ◽  
pp. 67-72
Author(s):  
Cintera Rahmagiarti ◽  
Silvia Tri Widyaningtyas ◽  
Budiman Bela
Keyword(s):  
Human Immunodeficiency Virus ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Internal Ribosomal Entry Site ◽  
Start Codon ◽  
Entry Site ◽  
Immunodeficiency Virus ◽  
Acquired Immunodeficiency ◽  
Ribosomal Entry ◽  
Hiv 1

Human immunodeficiency virus (HIV) is a virus that causes acquired immunodeficiency virus syndrome (AIDS). The HIV genome has a cap structure at 5’ and polyadenylation at 3’ on mRNA resulting in a translation initiation through scanning at 5'untranslated region (UTR). The Vpr protein produced during viral replication causes the 5'cap scanning to be inhibited so HIV-1 can directly recruit the ribosome at the start codon via internal ribosomal entry site (IRES). IRES activity is high at G2/M phase and highest expression in monocyte cell line (THP-1) and lymphocyte (HPB-ALL). The role of HIV IRES however, is not yet known in infection of nondividing cells by HIV-1. HIV-1 IRES and egfp from pcDNA5FRT/TO were amplified with PCR. The insert DNA (HIV-1 IRES_egfp) and pcDNA3.1(+) were digested with EcoRI and ApaI and then ligated. The verification was performed with PCR colonies, restriction verification, and sequencing. The size of insert DNA is 1067 bp while the vector is 5379 bp. E. coli transformed with DNA ligation produces 70 colonies, control of ligation produces 5 colonies, and negative control didn’t grow. 19 colonies contain recombinant DNA, restriction verification was of the appropriate size, and the sequence verification didn’t find any mutation. Therefore, the subcloning process pcDNA3.1_IRES HIV-1_egfp was successfully performed. Abstrak Human immunodeficiency virus (HIV) merupakan virus penyebab acquired immunodeficiency virus syndrome (AIDS). Genom HIV memiliki struktur cap di 5’ dan poliadenilasi di 3’ mRNA sehingga proses inisiasi translasi melalui pemindaian 5’cap pada struktur untranslated region (UTR) di 5’ mRNA HIV. Protein Vpr yang dihasilkan selama replikasi virus menyebabkan pemindaian melalui 5’cap terhambat sehingga HIV-1 dapat langsung merekrut ribosom pada kodon awal translasi melalui struktur internal ribosomal entry site (IRES). Aktivitas IRES tinggi pada fase G2/M dan ekspresi gen tinggi pada sel line monosit (THP-1) dan limfosit (HPB-ALL). Namun, peran IRES HIV-1 belum diketahui pada sel tidak membelah yang merupakan sel target pada infeksi HIV-1. DNA sekuen IRES HIV-1 dan egfp dari pcDNA5FRT/TO diamplifikasi dengan PCR. DNA sisipan (IRES HIV-1_egfp) dan pcDNA3.1(+) dipotong dengan EcoRI dan ApaI lalu DNA sisipan diligasi dengan pcDNA3.1(+). Verifikasi hasil klona dilakukan dengan PCR koloni, verifikasi restriksi, dan sekuensing. Restriksi DNA sisipan menghasilkan pita berukuran 1067 pb. Restriksi vektor plasmid menghasilkan pita berukuran 5379 pb. E.coli yang ditransformasi DNA ligasi menghasilkan 70 koloni, kontrol ligasi 5 koloni, dan kontrol negatif tidak tumbuh. 19 koloni terverifikasi mengandung DNA rekombinan, verifikasi restriksi memiliki ukuran sesuai, dan verifikasi sekuensing tidak terdapat perubahan basa. Oleh karena itu, proses subkloning pcDNA3.1_IRES HIV-1_egfp berhasil dilakukan.

scholarly journals DDX3 Participates in Translational Control of Inflammation Induced by Infections and Injuries

2018 ◽  
Vol 39 (1) ◽  
Author(s):  
Yu-Chang Ku ◽  
Min-Hua Lai ◽  
Chen-Chia Lo ◽  
Yi-Chuan Cheng ◽  
Jian-Tai Qiu ◽  
...  
Keyword(s):  
Translational Control ◽  
Fluorescent Protein ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Antibody Array ◽  
Transgenic Zebrafish ◽  
Pathway Enrichment Analysis ◽  
Translational Efficiency ◽  
Macrophage Migration ◽  
Target Mrnas

ABSTRACT Recent studies have suggested that DDX3 functions in antiviral innate immunity, but the underlying mechanism remains elusive. We previously identified target mRNAs whose translation is controlled by DDX3. Pathway enrichment analysis of these targets indicated that DDX3 is involved in various infections and inflammation. Using immunoblotting, we confirmed that PACT, STAT1, GNB2, Rac1, TAK1, and p38 mitogen-activated protein kinase (MAPK) proteins are downregulated by DDX3 knockdown in human monocytic THP-1 cells and epithelial HeLa cells. Polysome profiling revealed that DDX3 knockdown reduces the translational efficiency of target mRNAs. We further demonstrated DDX3-mediated translational control of target mRNAs by luciferase reporter assays. To examine the effects of DDX3 knockdown on macrophage migration and phagocytosis, we performed in vitro cell migration assay and flow cytometry analysis of the uptake of green fluorescent protein-expressing Escherichia coli in THP-1 cells. The DDX3 knockdown cells exhibited impaired macrophage migration and phagocytosis. Moreover, we used a human cytokine antibody array to identify the cytokines affected by DDX3 knockdown. Several chemokines were decreased considerably in DDX3 knockdown THP-1 cells after lipopolysaccharide or poly(I·C) stimulation. Lastly, we demonstrated that DDX3 is crucial for the recruitment of phagocytes to the site of inflammation in transgenic zebrafish.

scholarly journals The non-primate hepacivirus 5′ untranslated region possesses internal ribosomal entry site activity

2013 ◽  
Vol 94 (12) ◽  
pp. 2657-2663 ◽  
Author(s):  
Hazel Stewart ◽  
Cheryl Walter ◽  
Dale Jones ◽  
Sinead Lyons ◽  
Peter Simmonds ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Stem Loop ◽  
Long Range Interactions ◽  
Ribosomal Entry ◽  
Internal Translation ◽  
And Function

The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.

AlphaLISA-based high-throughput screening assay to measure levels of soluble amyloid precursor protein α

2014 ◽  
Vol 459 ◽  
pp. 24-30 ◽  
Author(s):  
Hongjie Wang ◽  
Adel Nefzi ◽  
Gregg B. Fields ◽  
Madepalli K. Lakshmana ◽  
Dmitriy Minond
Keyword(s):  
Amyloid Precursor Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Precursor Protein ◽  
Screening Assay ◽  
High Throughput Screening Assay
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