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Science 19 September 1986:
Vol. 233. no. 4770, pp. 1294 - 1299
DOI: 10.1126/science.3638792
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Articles

Science, Vol 233, Issue 4770, 1294-1299
Copyright © 1986 by American Association for the Advancement of Science

articles

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome

PJ Grabowski and PA Sharp

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.


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The Pre-mRNA 5' Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5' Splice Sites.
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The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing..
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Purification of the Spliceosome A-Complex and Its Visualization by Electron Microscopy.
E. Furman and D. G. Glitz (1995)
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Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP.
J. Crispino, B. Blencowe, and P. Sharp (1994)
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Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing..
R R Gontarek, M T McNally, and K Beemon (1993)
Genes & Dev. 7, 1926-1936
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A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals..
S Michaud and R Reed (1993)
Genes & Dev. 7, 1008-1020
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Interaction of the human autoantigen p150 with splicing snRNPs.
B. Blencowe, M Carmo-Fonseca, S. Behrens, R Luhrmann, and A. Lamond (1993)
J. Cell Sci. 105, 685-697
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Protein components specifically associated with prespliceosome and spliceosome complexes..
M Bennett, S Michaud, J Kingston, and R Reed (1992)
Genes & Dev. 6, 1986-2000
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Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing.
D. Wassarman and J. Steitz (1992)
Science 257, 1918-1925
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Association of U6 snRNA with the 5'-splice site region of pre-mRNA in the spliceosome..
H Sawa and Y Shimura (1992)
Genes & Dev. 6, 244-254
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An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway..
S Michaud and R Reed (1991)
Genes & Dev. 5, 2534-2546
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Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.
H. Kuo, F. Nasim, and P. Grabowski (1991)
Science 251, 1045-1050
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A Sequential splicing mechanism promotes selection of an optimal exon by repositioning a downstream 5' splice site in preprotachykinin pre-mRNA..
F H Nasim, P A Spears, H M Hoffmann, H C Kuo, and P J Grabowski (1990)
Genes & Dev. 4, 1172-1184
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The organization of 3' splice-site sequences in mammalian introns..
R Reed (1989)
Genes & Dev. 3, 2113-2123
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Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction.
M. Hentze, T. Rouault, J. Harford, and R. Klausner (1989)
Science 244, 357-359
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An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly.
S. Ruby and J Abelson (1988)
Science 242, 1028-1035
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Presplicing complex formation requires two proteins and U2 snRNP..
A Kramer (1988)
Genes & Dev. 2, 1155-1167
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Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing..
K K Nelson and M R Green (1988)
Genes & Dev. 2, 319-329
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Spliceosome assembly in yeast..
S C Cheng and J Abelson (1987)
Genes & Dev. 1, 1014-1027
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A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation..
A I Lamond, M M Konarska, and P A Sharp (1987)
Genes & Dev. 1, 532-543
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A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae..
B C Rymond, D D Torrey, and M Rosbash (1987)
Genes & Dev. 1, 238-246
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Splicing of yeast nuclear pre-mRNA in vitro requires a functional 40S spliceosome and several extrinsic factors..
R J Lin, A J Lustig, and J Abelson (1987)
Genes & Dev. 1, 7-18
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Splicing of messenger RNA precursors.
P. Sharp (1987)
Science 235, 766-771
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Splicing of Messenger RNA Precursors.
P.A. Sharp, M.M. Konarksa, P.J. Grabowski, A.I. Lamond, R. Marciniak, and S.R. Seiler (1987)
Cold Spring Harb Symp Quant Biol 52, 277-285
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Different Small Nuclear Ribonucleoprotein Particles Are Involved in Different Steps of Splicing Complex Formation.
D. Frendewey, A. Kramer, and W. Keller (1987)
Cold Spring Harb Symp Quant Biol 52, 287-298
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