McConnell T S, Steitz J A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, CT 06536, USA.
EMBO J. 2001 Jul 2;20(13):3577-86. doi: 10.1093/emboj/20.13.3577.
A photoactivatable azidophenacyl group has been introduced into seven positions in the backbone of the 11 nucleotide invariant loop of U5 snRNA. By reconstituting depleted splicing extracts with reassembled U5 snRNP particles, molecular neighbors were assessed as a function of splicing. All cross-links to the pre-mRNA mapped to the second nucleotide downstream of the 5' splice site, and formed most readily when the reactive group was at the phosphate between U5 positions 42 and 43 or 43 and 44. Both their kinetics of appearance and sensitivity to oligonucleotide inhibition suggest that these cross-links capture a late state in spliceosome assembly occurring immediately prior to the first step. A later forming, second cross-linked species is a splicing product of the first cross-link, suggesting that the U5 loop backbone maintains this position through the first step. The proximity of the U5 loop backbone to the intron's 5' end provides sufficient restrictions to develop a three-dimensional model for the arrangement of RNA components in the spliceosome during the first step of pre-mRNA splicing.
一个可光激活的叠氮苯甲酰基已被引入到U5 snRNA的11个核苷酸不变环主干的7个位置。通过用重新组装的U5 snRNP颗粒重构建耗尽的剪接提取物,根据剪接功能评估分子邻域。所有与前体mRNA的交联都定位在5'剪接位点下游的第二个核苷酸处,并且当反应基团位于U5位置42和43或43和44之间的磷酸处时最容易形成。它们出现的动力学和对寡核苷酸抑制的敏感性都表明,这些交联捕获了剪接体组装中紧接第一步之前发生的晚期状态。后来形成的第二个交联物种是第一个交联的剪接产物,这表明U5环主干在第一步中保持这个位置。U5环主干与内含子5'端的接近提供了足够的限制,从而能够建立一个三维模型,用于描述前体mRNA剪接第一步中剪接体中RNA成分的排列。