RNA



ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5′ splice site


RIZWANA K.  ALVI a1, METTE  LUND a2 and RAYMOND T.  O'KEEFE a1c1
a1 School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom
a2 Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark

Abstract

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5′ exon before the first step of splicing and with the 5′ and 3′ exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5′ and 3′ exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5′ exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5′ exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5′ splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5′ exon in two steps during its targeting to the 5′ splice site.

(Received March 20 2001)
(Accepted April 9 2001)


Key Words: 5′ splice site; pre-mRNA splicing; spliceosome; U5 snRNA; yeast.

Correspondence:
c1 Reprint requests to: Raymond T. O'Keefe, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom; e-mail: rokeefe@man.ac.uk.