Stoichiometry of the Sm proteins in yeast spliceosomal snRNPs supports theheptamer ring model of the core domain

Seven Sm proteins (B/B ', D1, D2, D3, E, F and G proteins) containing a com mon sequence motif form a globular core domain within the U1, U2, U5 and U4 /U6 spliceosomal snRNPs. Based on the crystal structure of two Sm protein d imers we have previously proposed a model of the snRNP core domain consisti ng of a ring of seven Sm proteins. This model postulates that there is only a single copy of each Sm protein in the core domain. In order to test this model we have determined the stoichiometry of the Sm proteins in yeast spl iceosomal snRNPs. We have constructed seven different yeast strains each of which produces one of the Sm proteins tagged with a calmodulin-binding pep tide (CBP). Further, each of these strains was transformed with one of seve n different plasmids coding for one of the seven Sm proteins tagged with pr otein A. When one Sm protein is expressed as a CBP-tagged protein from the chromosome and a second protein was produced with a protein A-tag from the plasmid, the protein A-tag was detected strongly in the fraction bound to c almodulin beads, demonstrating that two different tagged Sm proteins can be assembled into functional snRNPs. in contrast when the CBP and protein A-t agged forms of the same Sm protein were co-expressed, no protein A-tag was detectable in the fraction bound to calmodulin. These results indicate that there is only a single copy of each Sm protein in the spliceosomal snRNP c ore domain and therefore strongly support the heptamer ring model of the sp liceosomal snRNP core domain. (C) 2001 Academic Press.