探究依赖于 DEAD-box 解旋酶 Dbp2 的转录组范围的 RNA 结构变化。
Probing Transcriptome-Wide RNA Structural Changes Dependent on the DEAD-box Helicase Dbp2.
机构信息
Department of Biochemistry, Purdue University, West Lafayette, IN, USA.
Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA.
出版信息
Methods Mol Biol. 2021;2209:287-305. doi: 10.1007/978-1-0716-0935-4_18.
RNA helicases function in all aspects of RNA biology mainly through remodeling structures of RNA and RNA-protein (RNP) complexes. Among them, DEAD-box proteins form the largest family in eukaryotes and have been shown to remodel RNA/RNP structures and clamping of RNA-binding proteins, both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to promote RNA metabolism. Several methods have been developed to probe secondary and tertiary structures of specific transcripts or whole transcriptomes in vivo. In this chapter, we describe a protocol for identification of RNA structural changes that are dependent on a Saccharomyces cerevisiae DEAD-box helicase Dbp2. Experiments detailed here can be adapted to the study of other RNA helicases and identification of putative remodeling targets in vivo.
RNA 解旋酶在 RNA 生物学的各个方面发挥作用，主要通过重塑 RNA 和 RNA-蛋白质（RNP）复合物的结构。其中，DEAD 盒蛋白在真核生物中形成最大的家族，已被证明可以在体外和体内重塑 RNA/RNP 结构和 RNA 结合蛋白的夹闭。然而，对于这些酶中的大多数，还不清楚靶向哪些 RNA 以及它们在哪里调节 RNA/RNP 结构以促进 RNA 代谢。已经开发了几种方法来探测特定转录本或整个转录组的二级和三级结构。在本章中，我们描述了一种鉴定依赖于酿酒酵母 DEAD 盒解旋酶 Dbp2 的 RNA 结构变化的方法。这里详述的实验可以适用于其他 RNA 解旋酶的研究和体内推定重塑靶标的鉴定。
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