Anderson K, Moore M J
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
RNA. 2000 Jan;6(1):16-25. doi: 10.1017/s1355838200001862.
Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3' splice site AG is supplied by a different RNA molecule than that containing the 5' splice and branch sites. By varying the time during splicing reactions when the 3' splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3' splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.
在此，我们报告了一种用于人类剪接体进行外显子连接的体外分析系统的进一步特性，在该系统中，3' 剪接位点AG由一个与包含5' 剪接位点和分支位点的RNA分子不同的RNA分子提供。通过改变剪接反应过程中3' 剪接位点AG可供剪接机制使用的时间，我们表明AG识别直到套索形成后才需要发生。因此，某些哺乳动物内含子上剪接体形成和套索形成所需的早期AG识别事件对于外显子连接并非必需。消耗/回补研究和冷竞争物挑战实验表明，3' 剪接位点AG对外显子连接的确定需要NTP水解。由于它在物理和动力学上都将外显子连接与剪接体组装和套索形成解偶联，因此双分子系统将成为进一步对剪接第二步进行机制分析的有价值工具。
