Dissecting RNA-interference pathway with small molecules.
RNA interference (RNAi) is a process whereby short-interfering RNAs (siRNA) silence gene expression in a sequence-specific manner. We have screened a chemical library of substituted dihydropteridinones and identified a nontoxic, cell permeable, and reversible inhibitor of the RNAi pathway in human cells. Biochemical and fluorescence resonance-energy transfer experiments demonstrated that one of the compounds, named ATPA-18, inhibited siRNA unwinding that occurred within 6 hr of siRNA transfection. Extracts prepared from ATPA-18-treated cells also exhibited a decrease in target RNA cleavage by activated RNA-induced silencing complex (RISC*). Interestingly, when activated RISC*, which harbors unwound antisense siRNA, was treated with ATPA-18 in vitro, target RNA cleavage was not affected, indicating that this compound inhibited siRNA unwinding or steps upstream of unwinding in the RNAi pathway. Our results also establish the timing of siRNA unwinding and show that siRNA helicase activity is required for RNAi. ATPA-18 analogs will therefore provide a new class of small molecules for studying RNAi mechanisms in a variety of model organisms and deciphering in vivo genetic functions through reverse genetics.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Ya-Lin
YL
Chiu
Chimmanamada U
CU
Dinesh
Chia-ying
CY
Chu
Akbar
A
Ali
Kirk M
KM
Brown
Hong
H
Cao
Tariq M
TM
Rana
D002460:Cell Line; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D034622:RNA Interference / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism
C503963:ATPA18; D006575:Heterocyclic Compounds, 3-Ring; D007211:Indoles; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex