MicroRNAs (miRNAs) are 20-22 nt non-coding RNAs that regulate gene expression post transcriptionally via base pairing to complementary target mRNAs. They have fundamental importance for development and stress adaptation in plants and animals. Although a molecular frame work for miRNA biogenesis, degradation and action has been established, many aspects of this important gene regulatory pathway remain unknown. This project explores four main points. First, we propose to use genetic approaches to identify factors required for translational repression by miRNAs in plants. This mode of action was until recently thought to occur only exceptionally in plants. My post doctoral work showed that it occurs in many miRNA-target interactions. The mechanism remains unknown, however, leaving open a fertile area of investigation. Second, we wish to test specific hypotheses regarding the in vivo role of miRNA mediated endonucleolysis of mRNA targets. Long believed to serve exclusively as a degradation mechanism, we propose to test whether this process could have important functions in biogenesis of long non-coding RNA derived from mRNAs.Third, my postdoctoral work has provided unique material to use molecular genetics to explore pathways responsible for miRNA degradation, an aspect of miRNA biology that only now is emerging as being of major importance. Finally, our unpublished results show that plant miRNAs and their associated effector protein Argonaute (AGO) are associated with membranes and that membrane association is crucial for function. This is in line with similar data recently obtained from different animal systems. We propose to use genetic, biochemical and cell biological approaches to clarify to which membrane compartment AGO and miRNAs are associated, how they are recruited to this compartment, and what the precise function of membrane association is.These innovative approaches promise to give fundamental new insights into the inner workings of the pathway.