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

Published online by Cambridge University Press:  06 July 2001

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

Type
Research Article
Copyright
2001 RNA Society

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