S1). with an HSP40 dimer that transfers the client to HSP70-ATP and catalyzes HSP70 ATPase activity leading to formation of a high-affinity, ternary HSP70-ADP-client complex (13, 14). The co-chaperone Hop then mediates client transfer to HSP90 for conformational maturation together with HSP90 co-chaperones whose identities remain incompletely described (11, 15). Single-molecule observations of encounters between siRNA duplexes and Ago2 indicates that conformational changes required for RISC loading involve a similar chaperone assembly line, such that chaperone activity opens the conformation of unloaded Ago2 and extends the dwell time of siRNA duplexes on Ago2 to increase the frequency of Ago2-siRNA duplex encounters that result in RISC formation (16, 17). It is unclear whether other chaperone-assisted conformational changes are involved in functions of the mature loaded RISC, but the presence of HSP70 in Ago1-containing RISC purified using capture oligonucleotides complementary to a specific miRNA suggests that Ago-chaperone interactions may not be confined to the loading process (18). Early studies of human Ago2 showed that it is a peripheral membrane protein that associates with rough endoplasmic reticulum (RER) and Golgi membranes in a manner dependent on HSP90 activity (19, 20). More recent studies have confirmed AGO association with endomembrane compartments in plants and animals (21,C23), and have shown that at least three important AGO functions occur in association with membranes. First, mRNA target repression can occur at the RER. In (29). We previously showed that small RNA activity is defective and that membrane association of the main miRNA effector in plants, AGO1, is decreased in mutants with lesions in the key enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (21). 3-Hydroxy-3-methylglutaryl-CoA reductase inhibition or knockdown of other components of the mevalonate pathway in also led to defective TP-472 miRNA function (30). The mevalonate pathway produces a cytoplasmic pool of isopentenyldiphosphate that serves as a precursor for several essential lipids (31). These include sterols required for physicochemical properties of biomembranes (32), dolichol required for protein glycosylation (33), and prenyldiphosphate chains required for post-translational modification of proteins (34). Our previous study pointed to the relevance of sterols for miRNA activity in (21), FRP whereas Shi and Ruvkun (30) concluded that dolichol, and hence protein farnesyl and geranylgeranyl transferases are heterodimeric enzymes composed of the same -subunit (PLURIPETALA (PLP) in (21), we tested whether protein farnesylation could also play a role in small RNA function. We first introduced reporter systems for miR156 (41), miR171 (42), and miR403 activity into and analyzed reporter expression or activity in WT compared with and mutants. These tests did not reveal clear defects in miRNA function (Fig. S1). In several cases, however, mutation of miRNA pathway components does not lead to observable defects in miRNA function on their own, but create a sensitized background in which defects become clearly observable only when combined with other weak mutations in miRNA pathway factors. For example, mutants in the HSP90 co-chaperone SQN show weak miRNA-related defects on their own, but the importance TP-472 of SQN for miRNA activity is revealed by its spectacular genetic interaction with weak mutant alleles (8). Similarly, at low temperature, mutants in the AGO protein ALG-1 show weakly penetrant defects in developmental transitions controlled by the and miRNAs, but those phenotypes become strongly exacerbated upon mutation of components of the Golgi-associated retrograde protein complex that affects miRNA levels, including those of the family (43). To test for such synthetic interactions, we constructed two sets of TP-472 double mutants with ((44, 45)), and the second with the hypomorphic allele (46). In contrast to and single mutants, double mutants formed cup-shaped cotyledons, filament-like structures instead of flowers and trumpet-shaped leaves (Fig. 1double mutants than in either single mutant, but this trend was not general to all target genes of transcription factors repressed by miRNAs (Fig. 1was also detected, because double mutants were clearly smaller than single mutants, and were completely sterile in contrast to either single mutant (Fig. S2). Although these clear genetic interactions do not allow precise molecular conclusions on links between protein farnesylation and miRNA action to be drawn, they do support the implication of protein farnesylation in developmental functions linked to, or possibly controlled by, the miRNA pathway. Open in a separate window Figure 1. Farnesyl transferase mutants show defects related to.