Purification of the spliceosome A-complex and its visualization by electron microscopy.
Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.
Furman
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Department of Biological Chemistry, UCLA School of Medicine, University of California 90024-1737, USA.
Glitz
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DG
D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016914:Ribonuclease H / Q000494:pharmacology; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000302:isolation & purification; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000302:isolation & purification; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017381:Spliceosomes / Q000648:ultrastructure*
D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear; D017413:Ribonucleoprotein, U2 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D016914:Ribonuclease H