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Results: 11 to 12 of 12

Publication Record


siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE, Fesik SW, Shen Y
(2005) Nucleic Acids Res 33: 4527-35
MeSH Terms: Agammaglobulinaemia Tyrosine Kinase, Base Sequence, Cell Line, Down-Regulation, G-Protein-Coupled Receptor Kinase 4, Gene Library, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, RNA Interference, RNA, Messenger, RNA, Small Interfering, Transcription Factors
Show Abstract · Added March 5, 2014
A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.
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14 MeSH Terms
A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.
Rameh LE, Arvidsson Ak, Carraway KL, Couvillon AD, Rathbun G, Crompton A, VanRenterghem B, Czech MP, Ravichandran KS, Burakoff SJ, Wang DS, Chen CS, Cantley LC
(1997) J Biol Chem 272: 22059-66
MeSH Terms: Agammaglobulinaemia Tyrosine Kinase, Amino Acid Sequence, Animals, Binding Sites, Blood Proteins, Kinetics, Membrane Proteins, Molecular Sequence Data, Phosphatidylinositol 4,5-Diphosphate, Phosphatidylinositol Phosphates, Phosphoproteins, Phosphotyrosine, Protein Binding, Protein-Tyrosine Kinases, Proteins, Receptors, Steroid, Son of Sevenless Proteins, Spectrin
Show Abstract · Added November 26, 2018
Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.
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18 MeSH Terms