Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins - PubMed (original) (raw)
Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins
S W Stevens et al. Proc Natl Acad Sci U S A. 1999.
Abstract
The yeast U4/U6.U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6.U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6.U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6.U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.
Figures
Figure 1
Total RNA and protein eluted from affinity chromatography purification. (A) Total RNA from the Ni-NTA elution step. Ten micrograms of total snRNPs from the Ni-NTA eluate were phenol-extracted, and RNA was precipitated with ethanol. Resuspended RNA was run on a 5% polyacrylamide gel and was stained with silver. Positions of U1, U2, U4, U5L, U5S, and U6 snRNAs are shown. (B) Total protein from each step of the affinity purification. The organic phase of the phenol extractions from 10 μg of protein from the α-Py (lane 1) and Ni-NTA (lane 2) chromatography steps was precipitated with acetone, and recovered proteins were run on a 12% SDS/PAGE gel. Positions of the epitope-tagged SmD3 protein and the contaminant His4 are indicated.
Figure 2
Glycerol gradient purification of the U4/U6⋅U5 snRNP. (A) RNA extracted from fractions 22–27 from a 10–30% glycerol gradient was run on a 5% polyacrylamide gel and was stained with silver. Positions of the snRNAs are indicated. (B) Proteins associated with the U4/U6⋅U5 snRNP. Proteins from 64 glycerol gradient fractions were precipitated with acetone, were run on a 12% high TEMED SDS/PAGE gel, and were stained with Coomassie blue. Positions of the 24 stably associated U4/U6⋅U5 snRNP proteins are indicated.
Figure 3
Mass spectrometry microsequencing spectrum from a Brr2 peptide. (A) Representative spectrum from an ≈1,158-Da tryptic peptide derived from _Brr_2. (B) MS/MS mass spectrometry analysis of fragmented 1,158-Da peptide. Masses calculated for the resulting ions were correlated to the entire nonredundant protein database. The labeled mass peaks for the Y′′ ions represent the protein sequence PDISSD from the Brr2 protein corresponding to amino acids 958–963.
Figure 4
Snu13 is highly conserved from yeast to man. Sequences with high degrees of identity to budding yeast Snu13(sc) were found from Schizosaccharomyces pombe (Snu13sp), C. elegans (Snu13ce), and human (Snu13hs). Regions of homology also were found with budding yeast Nhp2. Sequences were aligned with the
pileup
program (GCG) and were aligned in
boxshade
(K. Hofman and M. Baron, Chemin des Boveresses, Lausanne, Switzerland). Identical amino acids are shaded black, with similar amino acids shaded gray.
References
- Staley J P, Guthrie C. Cell. 1998;92:315–326. - PubMed
- Vijayraghavan U, Company M, Abelson J. Genes Dev. 1989;3:1206–1216. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases