MicroRNAs (miRNAs) consist of a large family of evolutionarily conserved small regulatory RNAs. To accomplish their regulatory roles, one strand of the mature miRNA duplexes is incorporated into Argonaute (Ago) proteins to form miRNA-induced silencing complexes (miRISCs) in the cytoplasm, which repress the expression of partially or fully complementary target mRNAs (reviewed in refs 1
). It has now become clear that many mRNAs targeted by miRISCs undergo rapid decay in the cytoplasm 6
. The recognition of mRNAs by miRISCs through imperfect miRNA-mRNA base-pairing often results in rapid deadenylation of target mRNAs 10
. However, if miRNA-mRNA base-pairing is perfectly complementary, endonucleolytic cleavage of targeted transcripts ensues 14
Studies in D. melanogaster
S2 cells show that miRNA-mediated decay (miRMD) involves several components: GW182, the Ccr4-Not deadenylase complex, the Dcp1-Dcp2 decapping complex, and Ago1. It was proposed that Ago1 recruits the Ccr4–Not deadenylase complex and/or the decapping complex via GW182 to miRNA target mRNAs to promote their decay 17
. However, several key mechanistic issues of this model need to be directly addressed by kinetic studies. For example, is miRMD triggered by deadenylation or by decapping? Are deadenylation and decapping coordinated during miRMD? If so, how is this accomplished? Moreover, does miRISC directly recruit a deadenylase complex to promote deadenylation?
The mechanism of miRMD in mammalian cells is still somewhat unclear. In a cell-free system using HEK293F cell extracts, let-7 miRNA was shown to repress translation mainly by directing deadenylation 20
. Similarly, an in vivo study using a c-fos
inducible promoter system to monitor the kinetics of miRMD during the G0 to G1 transition showed that miRNAs modestly enhance deadenylation of target mRNAs 12
. However, the cause-and-effect relationship between deadenylation and decay of the RNA body was not directly addressed nor were the participating poly(A) nucleases identified. In addition, it is unclear whether decapping also plays a role in mammalian miRNA-mediated decay.
There are three paralogs of GW182 in mammals (reviewed in refs. 17
). The human members of this protein family are designated as trinucleotide repeat containing (TNRC)-6A, 6B, and 6C. All three TNRC6 proteins can interact with members of the Ago subfamily and are involved in miRNA-mediated mRNA repression in mammalian cells22
. Mammalian Ago proteins have a modestly conserved "m7G-cap-binding protein" motif (also known as the MID domain) required for miRNA-mediated translation repression 32
. When two conserved phenylalanines (F470 and F505) in the MID domain of human Ago2 were mutated to valines, the Ago2(F2V2) mutant protein was unable to repress translation 32
. It was suggested that the human Ago2(F2V2) lost the ability to repress translation due to its incapability to bind the 5’-cap 32
. However, the F2V2 mutant of Drosophila Ago1 (equivalent to Ago2 in mammals) was reported to retain the ability to bind the m7G–cap but could no longer interact with GW182 18
. Thus, it remains controversial as to why the F2V2 mutation impairs the mRNA silencing function of miRNAs.
In this study, we used a transcriptional pulse-chase approach to monitor the effect of inactivating individual deadenylases, the decapping enzyme Dcp2, or a combination of these enzymes on the deadenylation and decay kinetics of an mRNA targeted by let-7 in mouse NIH3T3 fibroblasts. Moreover, we combined a λ-N peptide/boxB hairpin mediated RNA-tethering approach with transcriptional pulse-chase to further address the roles of Ago and GW182 proteins in miRMD. Our results provide crucial new insights into the mechanism of miRMD.