Summary: MOLECULAR AND CELLULAR BIOLOGY, May 2003, p. 34423455 Vol. 23, No. 10
0270-7306/03/$08.00 0 DOI: 10.1128/MCB.23.10.34423455.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
The U1 snRNP Base Pairs with the 5 Splice Site within a
Hadar Malca, Noam Shomron, and Gil Ast*
Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University,
Ramat Aviv 69978, Tel Aviv, Israel
Received 4 November 2002/Returned for modification 23 December 2002/Accepted 13 February 2003
Recognition of the 5 splice site is an important step in mRNA splicing. To examine whether U1 approaches
the 5 splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system
in which a short RNA containing the 5 splice site sequence served as a substrate in a binding reaction. This
system allowed us to study the interactions of the snRNPs with the 5 splice site without the effect of other
cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal
snRNPs form a complex that specifically binds the 5 splice site through base pairing with the 5 end of U1.
This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5 splice site,
a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5 splice site are
base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs.
This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins.
The results argue that U1 can bind the 5 splice site in a mammalian preassembled penta-snRNP complex.