Mature microRNAs (miRNAs) are small RNAs of 21-24 bases that are emerging as important post-transcriptional regulators in many systems. The majority of miRNAs tested so far act by binding to a target mRNA, leading to translational repression or degradation of the transcript. Furthermore many known miRNA targets contain multiple binding sites for miRNAs, and it has been shown that miRNAs can act combinatorially to regulate mRNAs. While there are 116 annotated miRNAs in C. elegans, the function of the vast majority of these is unknown. However recent advances in computational prediction of miRNA targets suggests that 1000s of mRNAs are potentially regulated by miRNAs. We have developed a program (PicTar) that predicts miRNA binding sites in 3UTRs. PicTar assesses conservation of miRNA binding sites within 3UTRs across species, and it has been shown that incorporation of this filter significantly decreases false positives in output when compared to randomized sequences (Krek et al, 2005, Nature Genetics, in press). In addition PicTar can predict combinatorial binding of miRNAs to 3 UTRs. Despite these significant advances in predicting miRNA targets, there is no large-scale system for validating miRNA regulation in vivo as yet. We have sought to set up such a system, and present a high-throughput pipeline for validating miRNA targets. Using the Gateway recombination cloning system we are generating the 3UTR of the putative target transcript or a 3UTR in which the putative miRNA binding sites have been mutated. These reporters are driven by sequences upstream of the putative target gene. We are using these constructs to transform C. elegans, and thereby drive expression of the reporters in the tissues in which the predicted target transcript is active. By carrying out a series of comparative expression analyses in transgenic worms using these reporters we have been testing 30 novel predicted targets of the
let-7 and
lin-4 miRNAs.