Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Mol Cell. 2010 Aug 13;39(3):385-95. doi: 10.1016/j.molcel.2010.07.014.
To investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5' splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5' splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5' splice site cleavage, we found that Prp16 can associate with spliceosomes before 5' splice site cleavage, consistent with a role for Prp16 in proofreading 5' splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5' splice site cleavage and exon ligation share a common ATP-dependent framework.
为了探究准确的前体 mRNA 剪接的机制，我们开发了一种灵敏的体外分析方法，可检测 5' 剪接位点切割的校对。我们通过破坏催化中心的金属配体相互作用使剪接体失活，并发现当 DEAH 盒 ATP 酶 Prp16 失活时，这些剪接体虽然仍能催化 5' 剪接位点切割，但速率降低。虽然 Prp16 在 5' 剪接位点切割之后才促进真正底物的剪接，但我们发现 Prp16 可以在 5' 剪接位点切割之前与剪接体结合，这与 Prp16 在 5' 剪接位点切割的校对中起作用一致。我们证实 Prp16 介导的排斥是可逆的，需要下游的丢弃途径，我们发现该途径需要 DEAH 盒 ATP 酶 Prp43，即剪接体解体因子。这些数据表明剪接体能够区分慢底物，并且建立 5' 剪接位点切割和外显子连接保真度的机制共享一个共同的 ATP 依赖性框架。
