O'Keefe R T, Newman A J
MRC Laboratory of Molecular Biology, Cambridge, UK.
EMBO J. 1998 Jan 15;17(2):565-74. doi: 10.1093/emboj/17.2.565.
The U5 snRNA loop 1 interacts with the 5' exon before the first step of pre-mRNA splicing and with the 5' and 3' exons following the first step. These U5-exon interactions are proposed to hold the exons in the correct orientation for the second step of splicing. Reconstitution of U5 snRNPs in vitro indicated that U5 loop 1-5' exon interactions are not necessary for the first catalytic step of splicing but are critical for the second step in yeast spliceosomes. We systematically made deletion and insertion mutations in loop 1 then monitored splicing activity and loop-exon interactions by cross-linking. Single nucleotide deletions or insertions in loop 1 permitted both steps of splicing. Larger insertions or deletions allowed the first step but progressively inhibited the second step. Analysis of selected loop 1 insertions and deletions by cross-linking revealed that inhibition of the second catalytic step resulted from misalignment of the 5' and 3' exons. These data indicate that the size of loop 1 is critical for proper alignment of the exons for the second catalytic step of splicing and that the 3' exon is positioned on loop 1 independently of the 5' exon.
U5小核仁核糖核酸（snRNA）的环1在信使核糖核酸（pre-mRNA）剪接的第一步之前与5'外显子相互作用，并在第一步之后与5'和3'外显子相互作用。这些U5-外显子相互作用被认为能使外显子在剪接的第二步中保持正确的方向。体外重建U5小核核糖核蛋白颗粒（snRNPs）表明，U5环1与5'外显子的相互作用对于剪接的第一个催化步骤不是必需的，但对酵母剪接体中的第二步至关重要。我们系统地对环1进行缺失和插入突变，然后通过交联监测剪接活性和环-外显子相互作用。环1中的单核苷酸缺失或插入允许进行剪接的两个步骤。更大的插入或缺失允许第一步，但逐渐抑制第二步。通过交联分析选定的环1插入和缺失表明，第二个催化步骤的抑制是由5'和3'外显子的错位导致的。这些数据表明，环1的大小对于剪接第二个催化步骤中外显子的正确排列至关重要，并且3'外显子独立于5'外显子定位在环1上。
