Browsing by Subject "Small interfering RNA"

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The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase
(Public Library of Science, 2010-10-05) Lee, Heng-Chi; Aalto, Antti P.; Yang, Qiuying; Chang, Shwu-Shin; Huang, Guocun; Fisher, Daniel; Cha, Joonseok; Poranen, Minna M.; Bamford, Dennis H.; Liu, Yi
The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.
Hepatitis C Virus Induces E6AP-Dependent Degradation of the Retinoblastoma Protein
(Public Library of Science, 2007-09-28) Munakata, Tsubasa; Liang, Yuqiong; Kim, Seungtaek; McGivern, David R; Huibregtse, Jon; Nomoto, Akio; Lemon, Stanley M
Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.
Inhibition of Cell Proliferation by an Anti-EGFR Aptamer
(Public Library of Science, 2011-06-08) Li, Na; Nguyen, Hong Hanh; Byrom, Michelle; Ellington, Andrew D.
Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (Kd = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.
ISGylation and phosphorylation : two protein posttranslational modifications that play important roles in influenza A virus replication
(2008-08) Hsiang, Tien-ying, 1976-; Krug, Robert M.
Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions.
Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis.
(Public Library of Science, 2010-07-29) Tiwary, Richa; Yu, Weipin; Li, Jing; Park, Sook-Kyung; Sanders, Bob G.; Kline, Kimberly
Background -- α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings -- α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion -- Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.
Tunable, responsive nanoscale hydrogels for intracellular delivery of small interfering RNA
(2013-05) Liechty, William Blaine; Peppas, Nicholas A., 1948-; Alper, Hal; Contreras, Lydia; Ellison, Chris; Roy, Krishnendu
Responsive, polybasic nanoscale hydrogels were synthesized using photoemulsion polymerization. The nanoscale hydrogels (nanogels) are approximately 50 nm in diameter and consist of a pH-responsive poly(2-(diethylaminoethyl methacrylate)) core with a poly(ethylene glycol) corona. The nanogels undergo a volume phase transition in response to environmental pH, and possess tunable physicochemical properties based on the copolymer composition. Aqueous solution properties of the nanogels were investigated using dynamic light scattering, pyrene fluorescence spectroscopy, and zeta potential measurements. Nanogel-mediated disruption of biological membranes was investigated with erythrocytes, giant unilammelar vesicles, and live cells to evaluate the potential of these nanogels to act as endosomolytic carriers for intracellular delivery of small interfering RNA. Selective membrane disruption was achieved by increasing nanogel hydrophobicity via copolymerization with tert-butyl methacrylate (TBMA). Nanogels with TBMA possessed an additional advantage of increased cytocompatibility owing to their reduced cationic charge density. These nanogels are able to efficiently entrap siRNA in the nanogel core and enhance cellular internalization of siRNA in model cell lines. The cell-line dependent mechanism of nanogel internalization and uptake and intracellular distribution of fluorescent nanogel/siRNA complexes was investigated with imaging flow cytometry. Functional activity of delivered siRNA was assessed using gene silencing assays. Modifications to the polymer chemistry were also explored to enhance the biological activity. A disulfide-containing crosslinker was synthesized, characterized and incorporated into nanogels. This crosslinker enabled the rapid nanogel degradation in response to reductive environments. A functional phenylalanine-derivative monomer (MAPA) was synthesized and characterized to serve as a bio-inspired substitute for TBMA. Nanogels copolymerized with MAPA demonstrate comparable siRNA delivery efficiency to its TMBA analogue. The ability to exert control over physicochemical properties and biological interactions of these nanogels by tuning polymer composition is a facile strategy to tailor material properties for specific applications. The hydrophobically-modified nanogels possess great potential as delivery vehicles to enhance the cellular internalization and endosomal escape of siRNA cargoes.