An ATP-independent U2 small nuclear ribonucleoprotein particle/precursor mRNA complex requires both splice sites and the polypyrimidine tract.
A complex is formed upon incubation of a precursor mRNA (pre-mRNA) with HeLA cell nuclear extract in the absence of added ATP (-ATP complex). Pre-mRNAs with mutations in the 5' splice site, the 3' splice site, or the polypyrimidine tract did not form this complex. Once formed, the -ATP complex was stable to competition by excess pre-mRNA. The complex was shown to contain the U2 small nuclear ribonucleoprotein particle (snRNP) and was distinct from the previously described U2 snRNP/pre-mRNA complex, the prespliceosome. These complexes have different electrophoretic mobilities, ATP requirements, and sensitivities to mutations of the 5' splice site. Although U1 snRNP was not found in the -ATP complex, a requirement for the U1 snRNP was suggested by immunodepletion experiments. The possible implications for the study of spliceosome formation are discussed.
Jamison
S F
SF
Section of Cell Growth, Regulation and Oncogenesis, Duke University Medical Center, Durham, NC 27710.
Garcia-Blanco
M A
MA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D000255:Adenosine Triphosphate