High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking.
High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking.
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DOI：
10.1186/1471-2199-11-48
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发表时间：
2010-06-23
影响因子：
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通讯作者：
Williamson JR
中科院分区：
文献类型：
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作者：
Carmel AB;Wu J;Lehmann-Blount KA;Williamson JR
星星/GSG蛋白通过结合靶mRNA转录物的5 '或3' UTR中的共有位点来调节后生动物中的基因表达。由于星星结构域之间的高度同源性，大多数星星蛋白识别相似的RNA共有序列。以前，许多充分表征的星星蛋白的共有序列被定义为星星蛋白结合元件的六聚体序列，称为SBE。C.秀丽线虫GLD-1和小鼠Quaking（Qk-1）是两种代表性的星星蛋白，它们结合相似的共有六聚体，所述共有六聚体仅在某些位置的优选核苷酸同一性上不同。早期的报道也鉴定了位于靶RNA中典型共有六聚体上游或下游的部分共有元件，尽管这些序列对总体结合能的相对贡献仍然不太清楚。此外，最近从C.线虫被认为与GLD-1和Qk-1类似地结合靶RNA共有位点。在此，使用荧光偏振和凝胶迁移率变动测定的组合来证明星星-2与GLD-1和Qk-1类似的RNA共有序列结合。这些测定还用于进一步描述在各种RNA环境中每个六聚体共有核苷酸对GLD-1、Qk-1和星星-2的高亲和力结合的贡献。此外，还测量了在靶RNA中的典型六聚体的上游或下游插入额外的全部或部分共有元件的效果，以更好地定义由不同星星蛋白识别的序列元件和RNA结构。这里提出的结果表明，一个单一的六聚体共识是足够的高亲和力RNA结合的星星蛋白，上游或下游部分共识元素可能会改变结合亲和力取决于序列和间距。确定星星蛋白高亲和力RNA结合的一般要求将有助于促进体内新调控靶点的鉴定。
STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1. Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins. The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA binding by STAR proteins will help facilitate the identification of novel regulatory targets in vivo.