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。虽然越来越多的本地化RNA正在被发现，但这些事件的功能重要性还没有被很好地理解。我们正在专注于我们已经确定的一条定位路径，该路径针对一些RNA到细胞突起的末端。我们发现，该途径的一个重要组成部分是肿瘤抑制蛋白腺瘤性息肉病结肠(APC)，它的突变是大多数结直肠癌进展的起始事件。在核糖核蛋白复合体中，APC与RNA在细胞突起处结合，我们称之为APC-RNPs。在我们最近的工作中，我们发现这些RNA的本地化是有效的细胞迁移和入侵所必需的。此外，我们还发现细胞外基质的刚性促进了APC-RNPs的定位，并描述了其潜在的机制。具体地说，我们发现细胞外环境的僵硬增加促进了肌动球蛋白的收缩，这反过来又促进了一类特定类型的去酪氨酸微管的形成。这些稳定的微管是APC-RNPs在突起定位和/或锚定所必需的。鉴于细胞外环境的僵硬促进了转移行为的各个方面，我们目前正专注于测试我们所发现的机制是否涉及促进转移细胞的迁移。APC-RNPs的另一个成分是RNA结合蛋白FUS/TLS。FUS是一组含有Prion结构域的蛋白质，它们具有凝聚形成液体分离相的特性，这些分离相似乎是在细胞核和细胞质中形成各种无膜室的基础。在FUS的情况下，与肌萎缩侧索硬化症(ALS)相关的突变促进了其形成这种类型的细胞质内含物的倾向。我们一直致力于了解这些细胞质内含物的形成如何影响APC-RNPs的定位和功能。我们先前已经证明，FUS的ALS相关突变体错位了APC-RNP，并误导了它们的翻译。在我们最近的工作中，我们已经证明了FUS包裹体影响RNA定位的机制是间接的，并涉及到Kinesin-1马达蛋白的一个新功能。具体地说，我们发现Kinesin-1通过靶向微管蛋白羧肽酶活性而在促进变性微管的形成方面发挥了新的作用。在FUS包涵体的存在下，Kinesin-1mRNA和Kinesin-1蛋白都被隔离在FUS包涵体中。这防止了有效地形成降解的微管，导致APC-RNPs的错误定位。这些发现揭示了一种调节微管细胞骨架的新机制，这可能是ALS神经退行性变的一个促成因素。此外，我们还有兴趣研究这一新途径在含有FUS基因易位的癌症中的任何贡献。

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 is not well understood. We are focusing on a localization pathway that we have identified, which targets a number of RNAs to the tips of cellular protrusions. We have found that an important component of this pathway is the tumor-suppressor protein Adenomatous Polyposis Coli (APC) whose mutation is the initiating event in the progression of the majority of colorectal cancers. APC associates with RNAs at cellular protrusions in ribonucleoprotein complexes, which we term APC-RNPs. In our recent work we have found that localization of these RNAs is required for efficient cell migration and invasion. Furthermore, we have discovered that the stiffness of the extracellular matrix promotes localization of APC-RNPs and we have delineated the underlying mechanism. Specifically, we found that increased stiffness of the extracellular environment promotes actomyosin contractility, which in turn enhances formation of a specific class of detyrosinated microtubules. These stable microtubules are required for localization and/or anchoring of APC-RNPs at protrusions. Given that the stiffness of the extracellular environment promotes various aspects of metastatic behavior, we are currently focusing on testing whether the mechanism we have uncovered is involved in promoting migration of metastatic cells. An additional component of APC-RNPs is the RNA-binding protein FUS/TLS. FUS belongs to a group of prion-domain containing proteins which exhibit the property of condensing to form liquid-demixed phases, which appear to underlie the formation of various membrane-less compartments in the nucleus and cytoplasm. In the case of FUS, mutations linked to Amyotrophic Lateral Sclerosis (ALS) promote its propensity to form such types of cytoplasmic inclusions. We have been focusing on understanding how formation of these cytoplasmic inclusions impacts on localization and function of APC-RNPs. We have previously shown that ALS-associated mutants of FUS mislocalize APC-RNPs and misdirect their translation. In our more recent work we have shown that the mechanism through which FUS inclusions affect RNA localization is indirect and involves a novel function of the kinesin-1 motor protein. Specifically, we find that kinesin-1 has a novel role in promoting formation of detyrosinated microtubules through targeting the tubulin carboxypeptidase activity. In the presence of FUS inclusions, both the kinesin-1 mRNA and the kinesin-1 protein become sequestered within FUS inclusions. This prevents efficient formation of detyrosinated microtubules leading to mislocalization of APC-RNPs. These findings reveal a novel mechanism regulating the microtubule cytoskeleton which could be a contributing factor in ALS neurodegeneration. We are additionally interested in investigating any contribution of this novel pathway in cancers containing translocations of the FUS gene.