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Purification of the yeast U4/U6⋅U5 small nuclear ribonucleoprotein particle and identification of its proteins

Stevens, Scott W. and Abelson, John (1999) Purification of the yeast U4/U6⋅U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proceedings of the National Academy of Sciences of the United States of America, 96 (13). pp. 7226-7231. ISSN 0027-8424. PMCID PMC22060. https://resolver.caltech.edu/CaltechAUTHORS:20141125-134157867

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.96.13.7226 DOIArticle
http://www.pnas.org/content/96/13/7226PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC22060/PubMed CentralArticle
Additional Information:© 1999 National Academy of Sciences. Contributed by John Abelson, April 26, 1999. We thank John Woolford, Amy Kistler, and Christine Guthrie for providing strains and hybridomas. We are grateful to Tony Moreno, Mary Young, and Terry Lee at the City of Hope Beckman Research Institute for their excellent mass spectrometry work (supported by National Institutes of Health Grant CA33572) and to Manny Ares, Tracy Johnson, Stephen Rader, and Hong Li for critical reviewing the manuscript. S.W.S. was supported by a postdoctoral fellowship PF4447 provided by the American Cancer Society. This work was supported by National Institutes of Health Grant GM32627 to J.A. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
NIHCA33572
American Cancer SocietyPF4447
NIHGM32627
Subject Keywords:pre-mRNA splicing; mass spectrometry
Issue or Number:13
PubMed Central ID:PMC22060
Record Number:CaltechAUTHORS:20141125-134157867
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20141125-134157867
Official Citation:Purification of the yeast U4/U6⋅U5 small nuclear ribonucleoprotein particle and identification of its proteins PNAS 1999 96 (13) 7226-7231; doi:10.1073/pnas.96.13.7226
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52153
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:26 Nov 2014 20:06
Last Modified:03 Oct 2019 07:39

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