Kent Oliver A, MacMillan Andrew M
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Nat Struct Biol. 2002 Aug;9(8):576-81. doi: 10.1038/nsb822.
Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly/recruitment and suggest a mechanism for the formation of the final active site of the mature spliceosome.
真核生物中从前体mRNA（pre-mRNA）切除内含子需要剪接体核心部位底物内反应性功能的并列，剪接的两个化学步骤在此发生。尽管pre-mRNA、前体剪接体和剪接体因子之间的一系列相互作用已得到充分证实，但剪接机制组装的分子机制以及剪接底物组织的时间基础仍知之甚少。在这里，我们使用连接到pre-mRNA底物上的定向羟基自由基探针来绘制剪接体组装过程中pre-mRNA底物的结构。这些研究表明在剪接体组装/募集过程中保守的pre-mRNA序列有早期组织和接近，并提出了成熟剪接体最终活性位点形成的机制。
