Partial purification and properties of a pre-mRNA splicing activity.
Precursor RNA substrates for splicing reaction were synthesized in vitro from a plasmid DNA in which the early region 2 gene of adenovirus 2 was fused to an efficient bacteriophage promoter (Salmonella phage 6). Pre-mRNA splicing activity from nuclear extracts of MOPC-315 mouse myeloma cells was partially purified 108-fold by three chromatographic steps. The in vitro splicing reaction catalyzed by the partially purified fractions was efficient (60-80% substrate conversion) and accurate at the nucleotide level. The reaction occurred with crude or purified fractions without any detectable lag and nucleotides (ATP or GTP) were absolutely required. Monoclonal anti-Sm antibodies that quantitatively immunoprecipitate U1 small nuclear ribonucleoprotein particles totally inhibited the splicing activity of the purified fractions, indicating that U1 small nuclear RNPs had co-purified with the activity and were absolutely required for the splicing reaction.
DiMaria
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Kaltwasser
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Goldenberg
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics; D009694:Nucleic Acid Precursors / Q000302:isolation & purification*; D010957:Plasmids; D012322:RNA Precursors; D012326:RNA Splicing; D012333:RNA, Messenger / Q000302:isolation & purification*; D012261:Ribonucleoproteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear
D004279:DNA, Viral; D009694:Nucleic Acid Precursors; D012322:RNA Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate