Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro
Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro
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DOI：
10.1016/j.jmb.2007.11.041
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发表时间：
2008-02-01
影响因子：
5.6
通讯作者：
Lambowitz, Alan M.
中科院分区：
文献类型：
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作者：
Mohr, Georg;Del Campo, Mark;Lambowitz, Alan M.
粗糙脉孢菌中的 DEAD-box 蛋白 CYT-19 和酿酒酵母中的 Mss116p 是通用 RNA 伴侣，在剪接线粒体 I 组和 II 组内含子以及翻译激活中发挥作用。这两种蛋白均包含与 N 端和 C 端结构域相连的保守 ATP 依赖性 RNA 解旋酶核心区域，后者具有与许多其他 DEAD-box 蛋白类似的基本尾部。在 CYT-19 中，这种基本尾部被证明有助于非特异性 RNA 结合，有助于将核心解旋酶区域束缚到结构化 RNA 底物上。在这里，多重序列比对和二级结构预测表明 CYT-19 和 Mss116p 属于 DEAD-box 蛋白的不同亚组，其 C 端结构域在基本尾部之前有一个定义的扩展 a 螺旋区域。我们发现，Mss116p 预测的 a 螺旋区域中的突变或 C 端截断会强烈抑制 RNA 依赖性 ATP 酶活性，导致翻译激活和 RNA 剪接功能丧失。这些发现表明α-螺旋区域可以稳定和/或调节RNA解旋酶核心的活性。相比之下，仅去除基本尾部的截短会留下高RNA依赖性ATP酶活性，并且仅导致体内和体外翻译和RNA剪接效率的适度降低。生化分析表明，基本尾部的缺失会导致 I 组和 II 组内含子 RNA 的非特异性结合减弱，令人惊讶的是，还会损害饱和蛋白质浓度下的 RNA 解旋以及 RNA 解旋酶核心对单链 RNA 的核苷酸依赖性紧密结合。总之，我们的结果表明，Mss116p C 端结构域的两个子区域以不同的方式作用来支持和调节核心解旋酶区域的活性，核心解旋酶区域的 RNA 解旋活性对于翻译和 RNA 剪接功能都至关重要。 (C) 2007 Elsevier Ltd. 保留所有权利。
The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended a-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted a-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the a-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions. (C) 2007 Elsevier Ltd. All rights reserved.