Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.
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.
Gottschalk
A
A
European Molecular Biology Laboratory (EMBL), Protein and Peptide Group, Meyerhofstr. 1, D-69117 Heidelberg.
Neubauer
G
G
Banroques
J
J
Mann
M
M
Lührmann
R
R
Fabrizio
P
P
D005656:Fungal Proteins / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry; D011233:Precipitin Tests; D016601:RNA-Binding Proteins / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000032:analysis*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes
D005656:Fungal Proteins; D016601:RNA-Binding Proteins; C120617:RSE1 protein, S cerevisiae; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins