The SRm160/300 splicing coactivator is required for exon-enhancer function

The SRm160/300 splicing coactivator is required for exon-enhancer function

^*Banting and Best Department of Medical Research, University of Toronto, Toronto, ON M5G 1L6, Canada; and ^†Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Phillip A. Sharp

Abstract

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing “coactivator,” SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound “activators” and the snRNP machinery of the spliceosome.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Banting and Best Department of Medical Research, C. H. Best Institute, 112 College Street, Room 410, Toronto, ON M5G 1L6, Canada. e-mail: b.blencowe{at}utoronto.ca.
ABBREVIATIONS:

ESE,

exonic splicing enhancer;

snRNP,

small nuclear ribonucleoprotein particle;

snRNA,

small nuclear RNA;

SR,

serine-arginine;

SRm160/300,

complex containing the SR matrix proteins of 160 and 300 kDa;

RS domain,

C-terminal domain rich in serine and arginine residues;

U2AF,

U2 snRNP auxiliary factor;

U2AF-35kDa,

U2AF 35-kDa subunit