RNA Localization in flies and mammals: the contribution of translational silencing and mRNA Degradation factors / LSD

定位mRNA的翻译控制是调节特定细胞亚结构域中蛋白质表达的常见机制，并在许多过程中发挥重要作用，如轴形成、不对称细胞分裂、细胞运动性和神经元突触可塑性（1-3）。定位的mRNA通常在核糖核蛋白颗粒（RNP）中转运，并且必须被抑制直到RNA到达其最终目的地。这是通过存在于这些转运RNP中的翻译阻遏物分子，例如Bruno、CPEB、eIF 4AIII、FMRP、Staufen和ZBP 1来实现的。最近发现mRNA降解因子在mRNA定位中也起重要作用。例如，oskar（osk）mRNA运输到果蝇卵母细胞的后部需要DEAD盒RNA解旋酶Me 31 b/DDX-6（一种去帽激活剂）和去帽酶的DCP-1亚基（从mRNA中去除5 '帽以触发其降解）。这两种蛋白质与osk mRNA共定位在卵母细胞后部。此外，osk mRNA定位还依赖于外显子连接复合物（EJC）和Staufen，这两者都与哺乳动物的mRNA衰变有关。EJC必须与终止密码子下游的mRNA结合以触发无义介导的衰变（NMD），而Staufen 1将NMD因子Upf 1募集到特定的mRNA 3 '-UTR，从而诱导新形式的mRNA衰变。在哺乳动物中，Staufen 1是树突状mRNA转运复合物的组分。此外，DEAD盒蛋白DDX-6，哺乳动物的同源物的Me 31 b，被发现在驱动蛋白相关的RNA颗粒从大鼠脑中分离。osk mRNA定位也被armitage（armi）、spindle-E（sture-E）和maelstrom中的突变破坏，所有RISC组分介导siRNA依赖性RNA降解和miRNA依赖性翻译沉默。相比之下，Argonaute蛋白Aubergine（Aub）和Piwi一旦被定位，就需要有效的osk mRNA翻译，并且这两种蛋白质与mRNA一起积累在后部。这两种蛋白质已被证明与一类新的小RNA（称为重复相关小干扰RNA（rasiRNA））相关联，以抑制生殖系中转座因子的活性。然而，目前还不清楚与osk mRNA定位的Aub和Piwi是否与rasiRNAs结合，也不清楚后者是否在osk mRNA的翻译或降解中发挥任何作用。最后，最近的证据表明，在哺乳动物中，不仅RNA降解，而且翻译沉默与RNA转运相关，因为非编码RNA，如microRNA（miRNAs）和较长的调控RNA，如BC 1，可以抑制转运过程中mRNA的翻译（22-24）。据推测，这种miRNA引导的沉默发生在另一类RNP中，称为加工体（Pbodies），其是无脊椎动物和脊椎动物细胞中mRNA降解的主要位点，并且被抑制的mRNA甚至可以在特异性信号进入细胞质后从P-body释放以进一步翻译。这就提出了一个问题，即P体是否提供了一个平台，用于转运被抑制的RNA，并作为协调降解，翻译和定位的中心发挥作用。因此，我们想调查是否P-体组件参与mRNA运输解决以下具体问题：蛋白质成分的RNA降解途径和RISC复合物发挥直接作用，在本地化RNA在果蝇卵母细胞或哺乳动物神经元？小的非编码RNA在两个系统中的转运过程中沉默mRNA吗？定位的mRNA与P体在转运到目的地之前、期间或之后是否相关？Argonaute家族成员Aub和Piwi在果蝇卵母细胞osk mRNA定位和翻译中的分子功能是什么？哺乳动物Ago家族的单个成员，例如Ago 1 -5，是否与树突状RNP中的特定miRNA相关？

Translational control of localized mRNAs is a common mechanism for regulating protein expression in specific cellular subdomains and plays an important role in a number of processes, such as axes formation, asymmetric cell division, cell motility, and neuronal synaptic plasticity (1-3). Localized mRNAs are usually transported in ribonucleoprotein particles (RNPs) and must be translationally repressed until the RNA reaches its final destination. This is achieved by translational repressor molecules, e.g. Bruno, CPEB,eIF4AIII, FMRP, Staufen and ZBP1 that are present in these transport RNPs. It has recently emerged that mRNA degradation factors also play an essential role in mRNA localization. For example, the transport of oskar (osk) mRNA to the posterior of the Drosophila oocyte requires the DEAD-box RNA helicase Me31b/DDX-6, a decapping activator, and the Dcp-1 subunit of the decapping enzyme that removes the 5'-cap from mRNAs to trigger their degradation. Both proteins colocalize with osk mRNA at the oocyte posterior. Furthermore, osk mRNA localization also depends on the exon junction complex (EJC) and Staufen, both of which have been implicated in mRNA decay in mammals. The EJC must be bound to an mRNA downstream of a stop codon to trigger nonsense mediated decay (NMD), while Staufen 1 recruits the NMD factor Upf1 to specific mRNA 3'-UTRs and thereby induces a novel form of mRNA decay. In mammals, Staufen 1 is a component of dendritic mRNA transport complexes. Moreover, the DEAD-box protein DDX-6, the mammalian homolog of Me31b, is found in kinesin-associated RNA granules isolated from rat brain. osk mRNA localization is also disrupted by mutations in armitage (armi), spindle-E (spn-E) and maelstrom, all RISC components mediating siRNA-dependent RNA degradation and miRNA-dependent translational silencing. In contrast, the Argonaute proteins, Aubergine (Aub) and Piwi, are required for efficient osk mRNA translation once it is localized, and both proteins accumulate at the posterior with the mRNA. Both proteins have been shown to associate with a new class of small RNAs called repeat-associated small interfering RNAs (rasiRNAs) to repress the activity of transposable elements in the germline. However, it is unclear whether Aub and Piwi that localize with osk mRNA are bound to rasiRNAs, nor whether the latter play any role in osk mRNA translation or degradation.Finally, there is recent evidence that not only RNA degradation but also translational silencing is coupled to RNA transport in mammals, since non-coding RNAs, such as microRNAs (miRNAs) and longer regulatory RNAs, e.g. BC1, can repress translation of mRNAs during transport (22-24). It has been postulated that this miRNA-guided silencing occurs in another class of RNPs, called processing bodies (Pbodies),which are the major sites of mRNA degradation in both invertebrate and vertebrate cells , and that repressed mRNAs can even be released from P-bodies upon specific signals into the cytoplasm for further translation. This raises the question of whether P-bodies provide a platform for the transport of translationally repressed RNAs, and function as centers that co-ordinate degradation, translation and localization. We would therefore like to investigate whether P-body components are involved in mRNA transport by addressing the following specific questions: Do protein components of the RNA degradation pathway and the RISC complex play a direct role in localizing RNAs in Drosophila oocytes or mammalian neurons? Do small non-coding RNAs silence mRNA during their transport in both systems? Do localized mRNAs associate with P-bodies, before, during or after their transport to their destination? What is the molecular function of the Argonaute family members Aub and Piwi in osk mRNA localization and translation in Drosophila oocytes? Are individual members of the mammalian Ago family, e.g. Ago1-5, associated with specific miRNAs in dendritic RNPs?