Purification and electron microscopic visualization of functional human spliceosomes.
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.
Zhou
Zhaolan
Z
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Sim
Jeonggu
J
Griffith
Jack
J
Reed
Robin
R
D001483:Base Sequence; D002458:Cell Fractionation; D006367:HeLa Cells; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010316:Particle Size; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*
D012322:RNA Precursors