Recent studies have indicated that microRNAs (miRNAs) can reduce the levels of their target transcripts and the expression of the translated proteins. Elisa Izaurralde and colleagues now provide further evidence that Drosophila melanogaster miRNAs silence gene expression by these two mechanisms, which both require the processing (P)-body component GW182.

Izaurralde and co-workers compared the expression profiles of GW182-depleted cells with those of argonaute-1 (AGO1)-depleted cells (AGO1 mediates gene silencing by miRNAs in D. melanogaster). The profiles were strikingly similar, which indicates that these proteins regulate a common set of mRNAs and are therefore likely to function in the same pathway.

By transfecting cells with a GW182 fusion construct that binds with high affinity to a luciferase reporter mRNA target, the authors demonstrated that GW182 silences expression of the bound transcripts. In cells that were depleted of AGO1, GW182 still silenced reporter expression, bypassing the requirement for AGO1, which indicates that GW182 functions downstream of AGO1. The authors also identified the N-terminal domain of GW182 as the AGO1-binding region.

Tethering of GW182 to the reporter mRNA caused a marked reduction in the levels of reporter mRNA, which could not account fully for the observed decrease in luciferase activity. This therefore indicated that GW182-mediated silencing can occur by two mechanisms — repression of protein expression and mRNA degradation. By monitoring the levels of reporter mRNA over time in the presence of GW182, the authors noticed a decrease in the half-life of the mRNA. The mRNAs also shortened slightly over time, which implied that deadenylation had occurred. Further tethering assays revealed that the CCR4–NOT deadenylase complex was required for GW182-mediated mRNA degradation. The DCP1–DCP2 decapping complex was similarly required for mRNA decay by GW182.

The present findings place GW182 firmly in the miRNA-silencing pathway...

Using various reporter constructs that comprised endogenous D. melanogaster mRNA targets that are each inhibited by a corresponding miRNA, the authors showed that miRNAs trigger reductions in mRNA levels to different extents. As expected, target-mRNA levels were upregulated and expression levels increased in cells that were depleted of GW182. Silencing of one of the reporter transcripts occurred mainly at the translational level and, as a result, depletion of deadenylase or decapping components did not restore luciferase expression. It is currently unclear however how GW182 represses translation.

The present findings place GW182 firmly in the miRNA-silencing pathway; nevertheless, several questions remain open. Is miRNA-mediated translational repression the cause of mRNA degradation or are these two independent mechanisms? And what determines the degree of mRNA decay and the regulation of gene expression at the level of translation or mRNA stability?