Hostname: page-component-7c8c6479df-94d59 Total loading time: 0 Render date: 2024-03-28T18:32:01.101Z Has data issue: false hasContentIssue false

Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis

Published online by Cambridge University Press:  24 April 2002

MELISSA S. JURICA
Affiliation:
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA
LAWRENCE J. LICKLIDER
Affiliation:
Taplin Biological Mass Spectrometry Facility, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
STEVEN P. GYGI
Affiliation:
Taplin Biological Mass Spectrometry Facility, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA
NIKOLAUS GRIGORIEFF
Affiliation:
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA
MELISSA J. MOORE
Affiliation:
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA
Get access

Abstract

We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3′ splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 × 240 Å that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing.

Type
Research Article
Copyright
2002 RNA Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)