A novel splicing factor is an integral component of 200S large nuclear ribonucleoprotein (InRNP) particles.
In previous studies we have shown that nuclear transcripts of several pre-mRNAs can be released from nuclei of mammalian cells in the form of large nuclear ribonucleoprotein (InRNP) particles. By electron microscopy, these particles appeared as compact composite structures, 50 nm in diameter, which invariably sedimented at the 200S region in sucrose gradients. In order to identify putative protein splicing factors associated with the 200S InRNP particles, a panel of monoclonal antibodies directed against these particles were screened for their ability to inhibit splicing of pre-mRNA in vitro. In this study we have focused on a nuclear protein of 88 kd in molecular weight, which is an integral component of the InRNP complex and is recognized by monoclonal antibodies from a specific clone. This protein has been identified here as a novel splicing factor by, (i) antibody inhibition of splicing in vitro and (ii) depletion of splicing activity from HeLa cell nuclear extract after removing the 88 kd polypeptide by immunoadsorption, and complementation of the depleted activity with an affinity-purified 88 kd antigen. This splicing factor has further been shown to be required for the assembly of an active splicing complex.
Ast
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Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Goldblatt
D
D
Offen
D
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Sperling
J
J
Sperling
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008815:Mice, Inbred Strains / Q000276:immunology; D008854:Microscopy, Electron; D008970:Molecular Weight; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D012322:RNA Precursors; D012261:Ribonucleoproteins