X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X 
inactivation specific transcript (Xist). The mechanism of chromosome-wide 
silencing by Xist is poorly understood. While it is established that the 5' 
region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a 
conserved 26-mer sequence, is essential for Xist-mediated silencing, 
high-resolution structural information for the A-repeats is not available. Here, 
we report the three-dimensional solution structure of a 14-mer hairpin in the 5' 
region of a human A-repeat. This hairpin is remarkably stable and adopts a novel 
AUCG tetraloop fold, the integrity of which is required for silencing. We show 
that, contrary to previous predictions, the 3' region of single or tandem 
A-repeats mediates duplex formation in vitro. Significantly, mutations in this 
region disrupt the inter-repeat duplex formation in vitro and abrogate the 
silencing function of Xist A-repeats in vivo. Our data suggest that the complete 
A-repeat region may be stabilized by inter-repeat duplex formation and, as such, 
may provide a platform for multimerization and specific recognition of the AUCG 
tetraloops by trans-acting factors.