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首页> 外文期刊>Journal of Molecular Biology >ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo.
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ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo.

机译：DEAD-box蛋白Mss116p在II组内含子体内和体外剪接中的ATP依赖性作用。

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摘要

The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5gamma) primarily by stabilizing a folding intermediate. Here we compare three aI5gamma constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5gamma, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5gamma splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.
机译：酵母DEAD-box蛋白Mss116p在剪接线粒体I和II组内含子时起一般RNA伴侣的作用。对于其大多数功能，Mss116p被认为使用ATP依赖的RNA展开技术来促进RNA结构转变，但已建议主要通过稳定折叠中间体来协助II类内含子（aI5γ）的折叠。在这里，我们比较了三种aI5gamma构建体：一种具有长外显子，一种具有短外显子，以及一个缺乏外显子的核酶构建体。长的外显子导致剪接较慢，表明它们错误折叠和/或稳定了非天然内含子结构。尽管如此，所有三种构建体的Mss116p加速均取决于ATP，并受到损害RNA解旋的突变的抑制，表明存在类似的机制。剪接分析的结果以及将核酶折叠和催化分开的新的两阶段分析表明，Mss116p对所有三个构建体的最大折叠需要解开ATP依赖的RNA。仅最小核酶构建体的一个亚群，而不是包含外显子的构建体，ATP非依赖性激活才有意义。如一般RNA伴侣所期望的那样，Mss116p也可以破坏天然核酶，该酶​​在去除Mss116p后可以重新折叠。最后，使用仅含单个内含子aI5gamma的线粒体DNA酵母菌株，我们显示Mss116p突变体在体内促进剪接至与其残留的ATP依赖性RNA解旋活性相关的程度。在一起，我们的结果表明，尽管DEAD-box蛋白在RNA折叠中发挥多种作用，但Mss116p在aI5γ剪接中的生理功能包括对ATP依赖性局部展开的要求，从而允许将非功能性RNA结构转换为功能性RNA结构。

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