The role of GW182 proteins in microRNA-mediated gene silencing

MicroRNAs are endogenous approximately 21-nucleotide-long non-coding RNAs that act as post-transcriptional regulators of gene expression by base pairing to target mRNAs. Mature miRNAs form part of ribonucleoprotein complexes, called miRNA-induced silencing complexes (miRISCs), that contain Argonaute (AGO) and GW182 as core proteins. Drosophila melanogaster contains only one GW182 protein (DmGW182) but there are three GW182 paralogs, TNRC6A, TNRC6B, and TNRC6C, encoded in mammalian genomes. Proteins of the GW182 family play an important role in the execution of miRNA-mediated repression. However, the molecular mechanism of GW182-mediated repression is not entirely understood.
In order to get a more comprehensive understanding of the mechanism of miRNA-mediated repression, we studied the function of GW182 proteins using human HEK293 cells and Drosophila S2 cells as model systems. As a result of these investigations, we identified the C-terminal fragment of the human GW182 protein TNRC6C (CED) as a key region mediating miRNA-induced repression by interacting with PABP via its PAM2 motif and by recruiting the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes via conserved tryptophan-containing motifs (W-motifs).
In addition, tethering assays in HEK293 cells and Drosophila S2 cells revealed that the C-terminal regions of GW182 proteins are able to repress not only polyadenylated but also poly(A)-free mRNAs. Interestingly, the W-motifs which are essential for interaction of the CED with the CCR4-CAF1-NOT complex, were also required for the repression of poly(A)-free mRNAs by the tethered CEDs of human TNRC6C and DmGW182. Indeed, direct tethering of CCR4-CAF1-NOT complex components in HEK293 or S2 cells repressed not only polyadenylated but also poly(A)-free mRNAs and the RNA levels of poly(A)-free mRNAs were either not affected or only slightly reduced, indicating that the major part of the repression was due to inhibition of translation. Finally, repression of poly(A)-free mRNAs in Drosophila S2 cells by tethered DmGW182 or its CED depended on NOT1 but repression by tethered CAF1 or CNOT1 was independent of GW182, indicating that NOT1 acts downstream of GW182 in the repression of poly(A)-free mRNAs.
Taken together, these data indicate that recruitment of the CCR4-CAF1-NOT complex mediated by W-motifs of GW182 proteins, in addition to inducing deadenylation, also contributes to translational repression.