Purification and Electron Microscopic Visualization of Functional Human Spliceosomes

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2002 Sep 7

PMID 12215496

Citations 39

Authors

Zhaolan Zhou

Jeonggu Sim

Jack Griffith

Robin Reed

Affiliations

Soon will be listed here.

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.

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