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Plant microRNAs (miRNAs) affect only a small number of targets with high sequence complementarity, while animal miRNAs usually have hundreds of targets with limited complementarity. We used artificial miRNAs (amiRNAs) to determine whether the narrow action spectrum of natural plant miRNAs reflects only intrinsic properties of the plant miRNA machinery or whether it is also due to past selection against natural miRNAs with broader specificity. amiRNAs were designed to target individual genes or groups of endogenous genes. Like natural miRNAs, they had varying numbers of target mismatches. Previously determined parameters of target selection for natural miRNAs could accurately predict direct targets of amiRNAs. The specificity of amiRNAs, as deduced from genome-wide expression profiling, was as high as that of natural plant miRNAs, supporting the notion that extensive base pairing with targets is required for plant miRNA function. amiRNAs make an effective tool for specific gene silencing in plants, especially when several related, but not identical, target genes need to be downregulated. We demonstrate that amiRNAs are also active when expressed under tissue-specific or inducible promoters, with limited nonautonomous effects. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).[1]
Comprehensive analysis of gene function requires the detailed examination of mutant alleles. In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-of-function alleles for most annotated genes. However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications. Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes. In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available. Typically, gene function is reduced post-transcriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules. A recently introduced approach is the use of artificial microRNAs (amiRNAs). Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.[2]
