Double-stranded RNA (dsRNA) is produced within cells by several processes that have an important impact on cell physiology. Examples include mRNA processing, switching off gene expression by endogenous antisense RNA, and replication of RNA viruses. Cells have evolved a novel machinery for dealing with these molecules, a dsRNA specific activity that generates base-pair mismatches, and thus unwinds dsRNA, by converting many adenosine residues to inosinne. Although the in vivo role of this dsRNA adenosine deaminase or DRADSA has remained obscure since its discovery in 1987, it is most likely to affect the fate of the RNA duplex formed between sense and antisense RNAs and to interfere with the translation of the mRNAs modified by DRADA. Precise knowledge of the fate of dsRNA in vivo or the molecular mechanism for suppression of targeted genes translation is prerequisite for applicaiton of antisense RNA technology to develop therapeutics designed to block viral infection or to treat tumors. The goal of this project is to better understand the effect of DRADA on the antisense RNA strategy. We have recently succeeded in isolating DRADA clones. We will conduct in-depth investigation on the effect of the DRADA on the sense and antisense RNA duplex and its fate using DRADA expression constructs and purified recombinant DRADA proteins available in our laboratory.
Two specific aims are sought: 1. The effect of DRADA on the stability of the dsRNA will be investigated by transfecting a DRADA conditional expression construct into a cell line in which the expression of the antisense c-fos RNA can be manupulated conditionally. In addition, the effect of DRADA on the translation of the antisense RNA targeted endogenous c-fos mRNA will be investigated. 2. Using purifed recombinant DRADA proteins, we will examine in vitro whether DRADA will attack the RNA strand of RNA:DNA, RNA:phosphorothioate modified DNA, and RNA: O-methylated-RNA duplexes. By combining the informaiton from all of these experiments, we aim to increase our understanding of the effect of DRADA on dsRNA and, thereby, the success of the antisense RNA strategy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
3P01CA072765-02S1
Application #
6296149
Study Section
Project Start
1998-09-03
Project End
1999-06-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Kini, Hemant K; Kong, Jian; Liebhaber, Stephen A (2014) Cytoplasmic poly(A) binding protein C4 serves a critical role in erythroid differentiation. Mol Cell Biol 34:1300-9
Ji, Xinjun; Wan, Ji; Vishnu, Melanie et al. (2013) ?CP Poly(C) binding proteins act as global regulators of alternative polyadenylation. Mol Cell Biol 33:2560-73
Ji, Xinjun; Kong, Jian; Liebhaber, Stephen A (2011) An RNA-protein complex links enhanced nuclear 3' processing with cytoplasmic mRNA stabilization. EMBO J 30:2622-33
Jin, Shenghao; Zhao, Huiwu; Yi, Yan et al. (2010) c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis. J Clin Invest 120:593-606
Deleavey, Glen F; Watts, Jonathan K; Alain, Tommy et al. (2010) Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing. Nucleic Acids Res 38:4547-57
Waggoner, Shelly A; Johannes, Gregg J; Liebhaber, Stephen A (2009) Depletion of the poly(C)-binding proteins alphaCP1 and alphaCP2 from K562 cells leads to p53-independent induction of cyclin-dependent kinase inhibitor (CDKN1A) and G1 arrest. J Biol Chem 284:9039-49
Flagler, K; Alexeev, V; Pierce, E A et al. (2008) Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells. Gene Ther 15:1035-48
Pattanayak, Vikram; Gifford, Lida K; Lu, Ponzy et al. (2008) Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): caveats with respect to the use of ""stem-loop"" oligonucleotides as probes for mRNA folding. RNA 14:657-65
Tang, XinJing; Swaminathan, Jyothishmathi; Gewirtz, Alan M et al. (2008) Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides. Nucleic Acids Res 36:559-69
Silva, Ana Luisa; Ribeiro, Patricia; Inacio, Angela et al. (2008) Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay. RNA 14:563-76

Showing the most recent 10 out of 45 publications