P68 RNA Helicase Unwinds the Human let-7 MicroRNA Precursor Duplex and Is Required for let-7-directed Silencing of Gene Expression

MicroRNAs are short, single- stranded RNAs that arise from a transient precursor duplex. We have identified a novel activity in HeLa cell extracts that can unwind the let- 7 microRNA duplex. Using partially purified material, we have shown that microRNA helicase activity requires ATP and has a native molecular mass of similar to 68 kDa. Affinity purification of the unwinding activity revealed co- purification of P68 RNA helicase. Importantly, recombinant P68 RNA helicase was sufficient to unwind the let- 7 duplex. Moreover, like its native homolog, P68 RNA helicase did not unwind an analogous small interfering RNA duplex. We further showed that knockdown of P68 inhibited let- 7 microRNA function. From our data, we conclude that P68 RNA helicase is an essential component of the let- 7 microRNA pathway, and in conjunction with other factors, it may play a role in the loading of let- 7 microRNA into the silencing complex.