Gu, S. & Rossi, J. J. Uncoupling of RNAi from active translation in mammalian cells. RNA 11, 38-44

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.
RNA (Impact Factor: 4.94). 02/2005; 11(1):38-44. DOI: 10.1261/rna.7158605
Source: PubMed


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.

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Available from: Shuo Gu
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    • "Rules of such off-target effects provide us the design guidelines for allele-specific siRNA that can selectively silence certain SNPs causing disease without inhibiting the expression of corresponding wild-type alleles. Another matter of concern is the potency of siRNA, which can be affected by the differential integration of both strands of an siRNA duplex into the RISC [27], by the local structure of the target site [28] and by the siRNA target site locations within an mRNA [19], [29], [30]. Two reports have suggested that siRNAs targeting the 3′-UTR of transcripts could have higher potency than siRNAs interacting with sites located in the coding region [19], [29]. "
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    • "Furthermore, untranslated mRNAs are refractory to RNAi in Drosophila (Kennerdell et al., 2002). However, studies using the ferritin IRE-IRP system in mammalian cells indicate that active translation is not necessary for siRNA-mediated cleavage and RNAi is more sensitive in the absence of translation (Gu and Rossi, 2005). By examining the expression of a gfp reporter, whose expression is regulated by the RNAi pathway, we showed that ribosomal biogenesis components are required for RNAi, while various translation factors and tRNA synthetases may repress RNAi, indicating that different steps in protein synthesis may play a distinct role in regulating RNAi. "
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    • "Nonsense-mediated decay of mRNAs is mediated by an interaction between the RNA splicing apparatus and the proteins involved in translation (Maquat 2004), and it may be that interactions between the RNA splicing apparatus and RISC are important for directing transcripts to the SGS3/RDR6/DCL4 pathway. The fact that TAS2(+) and TAS1a(+) are noncoding transcripts may also be important because there is evidence in mammals that untranslated mRNAs are much more susceptible to RNAi than translated mRNAs (Gu and Rossi 2005). "
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