Impact of RNA structure on ZFP36L2 interaction with luteinizing hormone receptor mRNA
Impact of RNA structure on ZFP36L2 interaction with luteinizing hormone receptor mRNA
复制标题
DOI：
10.1261/rna.060467.116
复制
发表时间：
2017-08-01
期刊：
影响因子：
4.5
通讯作者：
Ramos, Silvia B. V.
中科院分区：
文献类型：
--
作者：
Ball, Christopher B.;Solem, Amanda C.;Ramos, Silvia B. V.
ZFP 36 L2（L2）使含AU富集元件（ARE）的转录物不稳定，并与女性生育力有关。我们已经证明，在小鼠促黄体激素受体mRNA的3' UTR内的三个推定战神中，只有一个ARE 2197（UAUUUAU）能够与L2相互作用。为了评估ARE 2197的结构元件是否可以解释这种独特的结合能力，我们对全长mLHR mRNA进行了全转录SHAPE-MaP（通过引物延伸-突变谱分析进行的选择性2'羟基酰化）。数据显示，功能性ARE 2197位于发夹环结构中，并且大多数核苷酸是高度反应性的。相比之下，每个非结合战神，2301和2444，只包含一个五聚体AUUUA;在ARE 2301中，大部分ARE序列是很难接近的。由于功能性mARE在人类中也被发现在序列水平上是保守的（ARE 2223），我们决定研究结合和结构是否也被保留。与小鼠类似，hLHR mRNA中只有一个ARE能够与L2结合;并且基于我们的SHAPE-MaP数据，它也位于发夹结构中。为了研究二级结构在结合中的作用，我们突变了两种功能战神中的特定核苷酸。在接近环的柔性茎区中的突变，其强制强碱基配对，显著降低L2结合亲和力;这证实了结构背景对于hARE 2223的L2识别是至关重要的。总的来说，我们的研究结果表明，最小的ARE序列的组合，放置在发夹环的ARE，和茎的灵活性介导高亲和力L2结合hLHR mRNA。
ZFP36L2 (L2) destabilizes AU-rich element (ARE)-containing transcripts and has been implicated in female fertility. We have shown that only one of three putative AREs within the 3' UTR of murine luteinizing hormone receptor mRNA, ARE2197 (UAUUUAU), is capable of interacting with L2. To assess whether structural elements of ARE2197 could explain this unique binding ability, we performed whole-transcript SHAPE-MaP (selective 2' hydroxyl acylation by primer extension-mutational profiling) of the full-length mLHR mRNA. The data revealed that the functional ARE2197 is located in a hairpin loop structure and most nucleotides are highly reactive. In contrast, each of the nonbinding AREs, 2301 and 2444, contains only a pentamer AUUUA; and in ARE2301 much of the ARE sequence is poorly accessible. Because the functional mARE was also found to be conserved in humans at the sequence level (ARE 2223), we decided to investigate whether binding and structure are also preserved. Similar to mouse, only one ARE in hLHR mRNA is capable of binding to L2; and it is also located in a hairpin structure, based on our SHAPE-MaP data. To investigate the role of secondary structure in the binding, we mutated specific nucleotides in both functional AREs. Mutations in the flexible stem region proximal to the loop that enforce strong basepairing, drastically reduced L2 binding affinity; this confirms that the structural context is critical for L2 recognition of hARE2223. Collectively, our results suggest that a combination of minimal ARE sequence, placement of the ARE in a hairpin loop, and stem flexibility mediate high-affinity L2 binding to hLHR mRNA.