In genomic imprinting, one of the two parental alleles of an autosomal gene is 
silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for 
a 400-kilobase region that contains three imprinted, maternally expressed 
protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion 
to be located in a sequence of 3.7 kilobases, which also contains the promoter 
for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is 
correlated with repression of all three genes on the paternal allele; however, 
Air RNA overlaps just one of these genes in an antisense orientation. Here we 
show, by inserting a polyadenylation signal that truncates 96% of the RNA 
transcript, that Air RNA is required for silencing. The truncated Air allele 
maintains imprinted expression and methylation of the Air promoter, but shows 
complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the 
paternal chromosome. Our results indicate that non-coding RNAs have an active 
role in genomic imprinting.