RNA



The role of U2AF35 and U2AF65 in enhancer-dependent splicing


BRENTON R.  GRAVELEY a1p1, KLEMENS J.  HERTEL a1p2 and TOM  MANIATIS a1c1
a1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA

Abstract

Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3′ splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3′ splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3′ splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3′ splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3′ splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.

(Received March 5 2001)
(Revised March 23 2001)
(Accepted April 3 2001)


Key Words: recruitment; splicing enhancer; SR protein; U2AF.

Correspondence:
c1 Reprint requests to: Tom Maniatis, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA; e-mail: maniatis@biohp.harvard.edu.
p1 Present address: Department of Genetics and Developmental Biology, University of Connecticut Health Center, School of Medicine, 263 Farmington Avenue, Farmington, Connecticut 06030-3301, USA.
p2 Present address: Department of Microbiology and Molecular Genetics, College of Medicine, B240 Medical Sciences I, University of California, Irvine, Irvine, California 92697-4025, USA.