Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6middle dotU5] tri-snRNP

Abstract

The 25S [U4/U6·U5] tri‐snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre‐mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri‐snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6·U5] tri‐snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri–snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm‐like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA‐binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre‐mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri‐snRNPs and the U2 snRNP are co‐precipitated with protein A‐tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2·U4/U6·U5] tetra‐snRNP complex.