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 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.
Salzman
David W
DW
Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 236 Farmington Avenue, Farmington, CT 06030, USA.
Shubert-Coleman
Jonathan
J
Furneaux
Henry
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D035683:MicroRNAs; D011994:Recombinant Proteins; C487931:mirnlet7 microRNA, human; D000255:Adenosine Triphosphate; D053487:DEAD-box RNA Helicases