Purification of Drosophila snRNPs and characterization of two populations of functional U1 particles.
U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans.
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Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D017124:Conserved Sequence; D004330:Drosophila; D006151:Guanosine / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012315:RNA Caps / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000302:isolation & purification* / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D000068103:Serine-Arginine Splicing Factors; D013045:Species Specificity
D009687:Nuclear Proteins; D012315:RNA Caps; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C490688:SNRNP70 protein, human; D006151:Guanosine; D000068103:Serine-Arginine Splicing Factors; C017778:N(2),N(2),7-trimethylguanosine; D017346:Protein Serine-Threonine Kinases