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Biochemical and genetic analyses of the U5, U6, and U4/U6[bull ]U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae

Published online by Cambridge University Press:  11 January 2002

SCOTT W. STEVENS ,
IMRE BARTA ,
HELEN Y. GE ,
ROGER E. MOORE ,
MARY K. YOUNG ,
TERRY D. LEE  and
JOHN ABELSON
SCOTT W. STEVENS
Affiliation:
California Institute of Technology, Division of Biology 147-75, Pasadena, California 91125, USA
IMRE BARTA
Affiliation:
California Institute of Technology, Division of Biology 147-75, Pasadena, California 91125, USA
HELEN Y. GE
Affiliation:
Division of Immunology, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
ROGER E. MOORE
Affiliation:
Division of Immunology, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
MARY K. YOUNG
Affiliation:
Division of Immunology, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
TERRY D. LEE
Affiliation:
Division of Immunology, Beckman Research Institute, City of Hope, Duarte, California 91010, USA
JOHN ABELSON
Affiliation:
California Institute of Technology, Division of Biology 147-75, Pasadena, California 91125, USA
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Abstract

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6[bull ]U5 snRNP. A novel yeast U5 and four novel yeast U4/U6[bull ]U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.

Keywords

affinity chromatographymass spectrometryspliceosome assembly
Type
Research Article
Information
RNA , Volume 7 , Issue 11 , November 2001 , pp. 1543 - 1553
Copyright
© 2001 RNA Society

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