Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome.
The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D012322:RNA Precursors; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000302:isolation & purification*; D019809:Streptavidin; D014981:Xenopus
D001426:Bacterial Proteins; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D001710:Biotin; D019809:Streptavidin