Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP.
Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.
Behzadnia
Nastaran
N
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
Hartmuth
Klaus
K
Will
Cindy L
CL
Lührmann
Reinhard
R
D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*
D012322:RNA Precursors; D017411:Ribonucleoproteins, Small Nuclear