Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells.
We describe the isolation and biochemical characterization of [U4/U6.U5] tri-snRNP complexes from HeLa cells under nondenaturing conditions using a monoclonal antibody reacting with the U5-specific 100-kD protein. We show that the [U4/U6.U5] complex contains five previously unobserved proteins with molecular masses of 90, 60, 27, 20, and 15.5 kD, in addition to the core proteins, common to the U4/U6, U5, U1, and U2 snRNPs, and the U5-specific proteins, as found in 20S U5 snRNPs. With approximately 20 distinct snRNP proteins the complexity of the [U4/U6.U5] tri-snRNP is surprising. One or more of the five proteins found exclusively in the 25S [U4/U6.U5] tri-snRNP appears to be involved in the assembly of the tri-snRNP complex, as, in an in vitro reconstitution assay, purified 20S U5 and 10S U4/U6 snRNPs formed stable 25S [U4/U6.U5] complexes only in the presence of the free tri-snRNP-specific proteins. The formation of the [U4/U6.U5] complex in vitro does not require ATP, and the stability of the purified tri-snRNP complex is not affected by ATP to a measurable extent. However, the native [U4/U6.U5] displays a kinase activity that is absent in isolated U5: A 52-kD protein present in both U5 and [U4/U6.U5] is phosphorylated only in the latter. The function of this phosphorylation is unclear thus far; it may be involved in the activation of [U4/U6.U5] in the spliceosome.
Behrens
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Institut für Molekularbiologie und Tumorforschung, Marburg, Germany.
Lührmann
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002467:Cell Nucleus / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis; D011494:Protein Kinases / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010455:Peptides; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D011494:Protein Kinases