Uncoupling of RNAi from active translation in mammalian cells.
Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.
Gu
Shuo
S
Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010, USA.
Rossi
John J
JJ
D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D035925:Iron-Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D035925:Iron-Regulatory Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins