The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11‐59K protein

U11 and U12 interact cooperatively with the 5′ splice site and branch site of pre‐mRNA as a stable preformed di‐snRNP complex, thereby bridging the 5′ and 3′ ends of the intron within the U12‐dependent prespliceosome. To identify proteins contributing to di‐snRNP formation and intron bridging, we investigated protein–protein and protein–RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12‐65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C‐terminal RRM and the U11‐associated 59K protein via its N‐terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3′ half of U12 forms an extended stem‐loop with a highly conserved seven‐nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12‐dependent, but not U2‐dependent, pre‐mRNA splicing. Taken together, these data suggest that U11/U12‐65K and U11‐59K contribute to di‐snRNP formation and intron bridging in the minor prespliceosome.