Abstract
Bulged nucleotides play a variety of important roles
in RNA structure and function, frequently forming tertiary
interactions and sometimes even participating in RNA catalysis.
In pre-mRNA splicing, the U2 snRNA base pairs with the
intron branchpoint sequence (BPS) to form a short RNA duplex
that contains a bulged adenosine that ultimately serves
as the nucleophile that attacks the 5′ splice site.
We have determined a 2.18-Å resolution crystal structure
of a self-complementary RNA designed to mimic the highly
conserved yeast (Saccharomyces cerevisiae) branchpoint
sequence (5′-UACUAACGUAGUA with
the BPS italicized and the branchsite adenosine underlined)
base paired with its complementary sequence from U2 snRNA.
The structure shows a nearly ideal A-form helix from which
two unpaired adenosines flip out. Although the adenosine
adjacent to the branchsite adenosine is the one bulged
out in the structure described here, either of these adenosines
can serve as the nucleophile in mammalian but not in yeast
pre-mRNA splicing. In addition, the packing of the bulged
RNA helices within the crystal reveals a novel RNA tertiary
interaction in which three RNA helices interact through
bulged adenosines in the absence of any divalent metal
ions.