Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast
Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast
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DOI：
10.1073/pnas.1315289111
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发表时间：
2013-12-24
影响因子：
11.1
通讯作者：
Niessing, Dierk
中科院分区：
文献类型：
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作者：
Niedner, Annika;Mueller, Marisa;Niessing, Dierk
mRNA的定向转运是真核生物的普遍特征，需要依赖运动的rna转运颗粒的组装。mrna的细胞质转运之前是信使前核糖核蛋白颗粒（mRNPs）的核组装。在出芽酵母中，HO 1 (ASH1) pre-mRNP的不对称合成已经开始共转录，并在核输出之前通过核核。ASH1 mRNA蛋白1 （Loc1p）的核仁定位是有效定位ASH1 mRNA的必要条件。免疫沉淀实验显示，Loc1p与ASH1转运复合物的其他组分形成复合物。然而，目前尚不清楚Loc1p是如何被招募到这个mRNP中的，以及为什么Loc1p对ASH1 mRNA的定位很重要。在这里，我们证明Loc1p与ASH1 mrna结合的swi5p依赖性HO表达蛋白2 （She2p）发生直接和特异性的相互作用。与单个蛋白相比，该复合物对携带定位元件的RNA具有更高的亲和力和特异性。它还稳定了She2p与ASH1 mRNA的短暂结合，表明Loc1p与She2p的协同mRNA结合是ASH1 mRNA定位所需的Loc1p核功能。核输出后，肌球蛋白结合的She3p加入ASH1 mRNP，与She2p和ASH1 mRNA形成高度特异性的复合物。因为Loc1p只存在于细胞核中，所以必须在输出之前或之后直接从复合物中去除。体外和体内实验表明，She2p和She3p的协同作用将Loc1p从ASH1复合体中置换出来，使游离的Loc1p迅速重新进入细胞核（ol）。综上所述，这些发现提示了一个有序的核组装和重组过程，以促进定位ASH1 mRNPs的成熟。
Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs.