Abstract
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 approximately 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.
Highlights
It is clear that the silencing activities of microRNAs are effected by a novel class of RNA-binding proteins called the Argonaute family [15,16,17,18]
The unwinding is likely facilitated by the cleavage of the passenger strand by Argonaute [25, 26], RNA helicase A has recently been implicated in loading of the guide strand of siRNA [27]
Neither the siRNA-directed nor the microRNA duplex-directed cleavage of a mRNA has been successfully reconstituted with recombinant proteins, and it is likely that additional factors are required for this step
Summary
B, the duplex let-7 substrate and its single stranded derivative (produced by heat penetration) as analyzed by native gel electrophoresis. C, 32P-labeled let-7 duplex was incubated with increasing amounts of crude extract made from. Reactions were deproteinized and analyzed for the presence of single-stranded RNA using native polyacrylamide gel electrophoresis. The asterisk indicates the addition of heat-inactivated extract. D, crude HeLa cell extract was fractionated using a Sephadex S-200 column. Fractions (2 l) were assayed for unwinding activity, using 32P-labeled let-7 duplex. E, 32P-labeled let-7 duplex was assayed for unwinding activity, using fraction 20 from the Sephadex S-200 column (2 g) in the absence or presence of different nucleoside cofactors (as indicated). The asterisk indicates a reaction containing a heat-inactivated Sephadex fraction.
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