Regulation and functional effects of localized RNAs

大量的RNA不是弥漫地分布在细胞质中，而是活跃地转运到各个亚细胞部位。在到达最终目的地后，本地化的RNA被翻译，从而指导当地的蛋白质生产。虽然正在确定越来越多的本地化RNA，但这些事件的功能重要性以及上述模型的总体适用性还没有得到很好的理解。我们正在专注于我们已经确定的一条定位途径，它以一些RNA为细胞突起的目标。这一途径之所以令人感兴趣，是因为它针对的是编码与各种类型癌症进展有关的因子的RNA。此外，正如我们已经发现的，它受疾病相关蛋白的调控，例如APC(腺瘤性息肉病结肠)，一种肿瘤抑制蛋白，其缺失是大多数结直肠肿瘤发生的基础，以及RNA结合蛋白FUS，一种在肌萎缩侧索硬化症病例中突变的蛋白。我们的总目标是：a)了解RNA在细胞突起定位的基本机制，b)了解其定位的功能结果，无论是作为一个组还是在单个转录本水平上，以及c)建立适当的模型系统和工具来研究生理和病理相关环境中的RNA定位事件。关于潜在的机制，我们已经揭示了RNA在突起的定位需要一个特定的修饰微管子集，在这个过程中，与神经退变相关的病理包裹体会破坏这个过程。我们进一步证明了RNA在突起的定位受到细胞外环境的机械性质的调节，并探讨了这种现象的分子基础。最后，我们发现了一个有趣的发现，与流行的模型相反，定位于突起的RNA的翻译不受它们在细胞质中的位置的影响，而是与特定的外周细胞过程相协调，在延伸突起时被激活，在突起回缩时被沉默。这种协调是动态的，并与RNA物理状态的变化有关。关于突起定位的RNA的功能作用，我们已经证明它们的定位对于有效的细胞迁移是重要的。我们已经设计出了破坏特定的单个RNA的定位的方法，并已经证明，仅干扰一个RNA的定位就足以在2D和3D系统中导致显著的迁移缺陷，以及减少癌细胞的集体侵袭。我们正在优化我们目前的体内递送方法，以进一步测试突起定位的RNA在肿瘤转移动物模型中的作用。除了迁移细胞外，我们最近的研究表明，依赖于APC的RNA也定位于非侵袭性的上皮细胞，在那里它们集中在基底表面。因此，另一条独立的研究路线是探索碱基定位的APC依赖的RNA在2D或3D上皮系统中的功能作用。鉴于APC在肠上皮中作为肿瘤发生的启动者的作用，我们相信这些研究可能揭示与肿瘤形成的初始阶段相关的局部RNA的作用。

A large number of RNAs are not diffusely distributed in the cytoplasm, but are actively transported to various subcellular sites. After reaching their final destinations, localized RNAs are translated, thus directing local protein production. While increasing numbers of localized RNAs are being identified, the functional importance of these events, as well as the overall applicability of the above model, is not well understood. We are focusing on a localization pathway that we have identified, which targets a number of RNAs to cellular protrusions. This pathway is of interest because it targets RNAs encoding factors that have been implicated in progression of various types of cancers. Furthermore, as we have found, it is regulated by disease-associated proteins, such as APC (Adenomatous Polyposis Coli), a tumor-suppressor protein whose loss underlies the initiation of most colorectal tumors, as well as by the RNA-binding protein FUS, a protein mutated in cases of Amyotrophic Lateral Sclerosis. Our overall goals have been to: a) understand the basic mechanisms underlying localization of RNAs at cell protrusions, b) understand the functional outcomes of their localization, either as a group or at the level of individual transcripts and c) set up appropriate model systems and tools to study RNA localization events in physiologically and pathologically relevant settings. With regards to the underlying mechanisms, we have revealed that localization of RNAs at protrusions requires a specific subset of modified microtubules in a process that is disrupted by pathologic inclusions associated with neurodegeneration. We have further shown that RNA localization at protrusions is modulated by the mechanical properties of the extracellular environment and have probed into the molecular basis of this phenomenon. Finally, we have made the intriguing discovery that, contrary to the prevailing model, translation of protrusion-localized RNAs is not affected by their position in the cytoplasm, but is rather coordinated with specific peripheral cellular processes, being activated at extending protrusions and silenced upon protrusion retraction. This coordination is dynamic and is coupled to a change in the physical state of the RNA. With regards to the functional roles of protrusion-localized RNAs, we have shown that their localization is important for efficient cell migration. We have devised ways to disrupt localization of specific, individual RNAs and have shown that disrupting the localization of just one RNA is sufficient to cause significant migration defects in 2D and 3D systems, as well as to reduce collective invasion of cancer cells. We are optimizing our current approaches for in vivo delivery to further test the roles of protrusion-localized RNAs in animal models of tumor metastasis. Apart from migrating cells, our recent studies have revealed that APC-dependent RNAs are also localized in non-invasive, epithelial cells, where they are concentrated at the basal surface. A separate line of investigation is thus exploring the functional roles of basally localized APC-dependent RNAs in 2D or 3D epithelial systems. Given the role of APC as an initiator of tumorigenesis in the intestinal epithelium, we believe these studies might reveal roles of localized RNAs relevant to the initial stages of tumor formation.