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  • Title: Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP.
    Author: Behzadnia N, Hartmuth K, Will CL, Lührmann R.
    Journal: RNA; 2006 Sep; 12(9):1738-46. PubMed ID: 16880538.
    Abstract:
    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.
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