RESUMO
Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.
Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/análise , Spliceossomos/química , Animais , Linhagem Celular , Galinhas/metabolismo , Ciclofilinas/análise , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/análise , Humanos , Espectrometria de Massas , Proteínas Nucleares/análise , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteômica , RNA Helicases/análise , Precursores de RNA/isolamento & purificação , RNA Mensageiro/isolamento & purificação , RNA Nuclear Pequeno/análise , RNA Nuclear Pequeno/isolamento & purificação , Proteínas de Ligação a RNA/análise , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/análise , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Fatores de Processamento de Serina-ArgininaRESUMO
Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.
