Published online 8 September 2004
Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved
Structural requirements for pre-microRNA binding and nuclear export by Exportin 5
1 Department of Molecular Genetics and Microbiology and 2 Howard Hughes Medical Institute, Box 3025, Duke University Medical Center, Durham, NC 27710, USA
* To whom correspondence should be addressed. Tel: +1 919 684 3369; Fax: +1 919 681 8979; Email: culle002{at}mc.duke.edu
Received July 16, 2004; Revised and Accepted August 25, 2004
The biogenesis and function of mature human microRNAs is dependent on the nuclear export of pre-microRNA precursors by Exportin 5 (Exp5). The precursor for the human miR-30 microRNA, which is a 63 nt long RNA hairpin bearing a 2 nt 3' overhang, forms a specific complex with Exp5 and the Ran-GTP cofactor. Here, we have examined the structural requirements for pre-microRNA binding by Exp5. Our data indicate that pre-miR-30 binding requires an RNA stem of >16 bp and is facilitated by a 3' overhang. Although a blunt-ended derivative of the pre-miR-30 stem–loop remained capable of binding Exp5, 5' overhangs were inhibitory. miR-30 variants that had lost the ability to bind Exp5 effectively were not efficiently exported from the nucleus and were also expressed at reduced levels. Furthermore, formation of a pre-microRNA/Exp5/Ran-GTP complex inhibited exonucleolytic digestion of the pre-miRNA in vitro. Together, these data demonstrate that pre-microRNA binding by Exp5 involves recognition of almost all of the RNA hairpin, with the exception of the terminal loop. Moreover, these results argue that Exp5 binding not only mediates pre-microRNA nuclear export but also prevents nuclear pre-microRNA degradation.
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