Capturing splicing complexes to study structure and mechanism.
At its most basic level, pre-mRNA splicing can be described as two coordinated nuclease reactions that cleave an intron at either end and result in ligation of the flanking exons. The fact that these reactions are catalyzed by a approximately 3-MDa behemoth of protein and RNA (the spliceosome) challenges most biochemical and structural approaches currently used to characterize lesser-sized enzymes. In addition to this molecular complexity, the highly dynamic nature of splicing complexes provides additional hurdles for mechanistic studies or three-dimensional structure determination. Thus, the methods used to study the spliceosome often probe individual properties of the machine, but no complete, high-resolution picture of splicing catalysis has yet emerged. To facilitate biochemical and structural studies of native splicing complexes, we recently described purification of the catalytic form of the spliceosome (known as C complex). This native complex is suitable for electron microscopic structure determination by single-particle methods. In this paper, we describe the purification in detail and discuss additional methods for trapping and analyzing other splicing complexes.
Jurica
Melissa S
MS
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Moore
Melissa J
MJ
D008854:Microscopy, Electron; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016914:Ribonuclease H / Q000494:pharmacology; D017381:Spliceosomes / Q000502:physiology / Q000648:ultrastructure
D012322:RNA Precursors; D016914:Ribonuclease H