Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP
Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP
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DOI：
10.1261/rna.120606
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发表时间：
2006-09-01
期刊：
影响因子：
4.5
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Behzadnia, Nastaran;Hartmuth, Klaus;Luehrmann, Reinhard
目前存在两种不同的剪接体组装途径模型，即传统模型，其中剪接体 snRNP 以逐步、有序的方式与前 mRNA 结合，以及全剪接体模型，其中所有剪接体 snRNP 预组装成五-snRNP 复合物。在这里，我们测试了仅包含与前体 mRNA 结合的 U1 和 U2 snRNP 的剪接体 A 复合物是否是一种功能性的、真正的组装中间体。值得注意的是，从去除 U4/U6 snRNP 的核提取物中亲和纯化的 A 复合物（因此无法形成五-snRNP）支持去除 U2 snRNP 的核提取物中的前 mRNA 剪接，而裸露的前 mRNA 则不支持。纯化的 A 复合物和裸露的前 mRNA 的混合实验还证实，在这些条件下，A 复合物不会从头形成。因此，我们的研究表明，全剪接体的形成并不是产生催化活性剪接体的先决条件，并且至少在体外，U1和U2 snRNP可以在功能上先于tri-snRNP并独立于tri-snRNP与pre-mRNA相关联。分离功能性剪接体 A 复合物的能力为使用纯化组分详细研究后续剪接体组装步骤铺平了道路。
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.