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Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12203-7. Epub 2002 Sep 5.

Purification and electron microscopic visualization of functional human spliceosomes.

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  • 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

Abstract

Pre-mRNA splicing takes place in a large and highly dynamic complex known as the spliceosome. Here we report the optimization of a maltose-binding protein (MBP) affinity-purification method to isolate functional spliceosomes for electron microscopic analysis. Visualization of the spliceosome preparations revealed distinct 40-60 nm particles. Immunogold-conjugated antibodies to spliceosome components specifically label these particles, which are eliminated by treatment with either RNase or protease. Moreover, spliceosomes assembled on two different pre-mRNAs are indistinguishable. This first visualization of purified functional spliceosomes assembled in vitro reveals striking structural features, including one or more central cavities and multiple elongate lobes.

PMID:
12215496
[PubMed - indexed for MEDLINE]
PMCID:
PMC129422
Free PMC Article

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