ABSTRACT
Gene silencing is an important tool in the study of gene function. Virus-induced gene silencing (VIGS) and hairpin RNA interference (hpRNAi), both of which rely on small interfering RNAs, together with artificial microRNAs (amiRNA), are amongst the most popular methods for reduction of gene activity in plants. However, all three approaches have limitations. Here, we introduce miRNA-induced gene silencing (MIGS). This method exploits a special 22-nucleotide miRNA of Arabidopsis thaliana, miR173, which can trigger production of another class of small RNAs called trans-acting small interfering RNAs (tasiRNAs). We show that fusion of gene fragments to an upstream miR173 target site is sufficient for effective silencing of the corresponding endogenous gene. MIGS can be reliably used for the knockdown of a single gene or of multiple unrelated genes. In addition, we show that MIGS can be applied to other species by co-expression of miR173.
Subject(s)
Arabidopsis/genetics , Gene Expression Regulation, Plant/genetics , Gene Knockdown Techniques/methods , Gene Silencing , MicroRNAs/genetics , RNA, Small Interfering/genetics , Flowers/genetics , Gene Fusion , Genomics , Mutation , Plants, Genetically Modified , Plasmids/genetics , RNA Interference , RNA, Plant/genetics , Nicotiana/genetics , Transcriptional Activation/geneticsABSTRACT
One mechanism for the origin of new plant microRNAs (miRNAs) is from inverted duplications of transcribed genes. However, even though many young MIRNA genes have recently been identified in Arabidopsis thaliana, only a subset shows evidence for having evolved by this route. We propose that the hundreds of thousands of partially self-complementary foldback sequences found in a typical plant genome provide an alternative path for miRNA evolution. Our genome-wide analyses of young MIRNA genes suggest that some arose from DNA that either has self-complementarity by chance or that represents a highly eroded inverted duplication. These observations are compatible with the idea that, following capture of transcriptional regulatory sequences, random foldbacks can occasionally spawn new miRNAs. Subsequent stabilization through coevolution with initially fortuitous targets may lead to fixation of a small subset of these proto-miRNA genes.
