Regulation and functional effects of localized RNAs

该研究计划侧重于定位RNA的作用，即针对特定亚细胞区域的RNA，以及局部RNA翻译在细胞迁移和癌症侵袭过程中的后果。这是一个研究明显不足的领域。我的团队的研究引领了对定位于非神经元迁移细胞突起的RNA的研究。多年来，我们在用于运输RNA到突起并调节其翻译的潜在分子机制；局部RNA翻译对蛋白质功能的影响；以及这些事件与癌症侵袭的相关性方面做出了许多贡献。我们已经开发了在2D和3D系统中研究突出局部RNA的方法，重点是成像以及产生无偏见的图像分析和量化方法(Stueland等人，2019年)。我们已经证明，RNA在突起的定位至少通过两条不同的途径进行，这两条途径取决于不同的蛋白质因素，并受细胞骨架的机械性能的不同调节(Wang等人，2017)。我们的大部分工作都集中在70个RNA的子集上，这些RNA需要肿瘤抑制因子APC进行定位。我们已经表明，依赖于APC的RNA的本地化对于2D和3D环境中的迁移是重要的。我们已经确定了在突起引导积累的顺式RNA元件；与这些RNA相关并控制其运输或翻译的蛋白质因子；以及扰乱这些过程的疾病突变(Mili等人，2008年；Yasuda等人，2013年；Yasuda等人，2017；Moissoglu，Pichon等人，提交中)。我们进一步揭示了控制局部转录本的新机制，表明APC依赖的RNA需要降解的微管，微管的形成受到细胞外基质的刚性、机械张力和肌球蛋白收缩能力的促进(Wang等人，2017)。我们发现了一种对本地化转录本进行翻译调控的新模式。使用最先进的方法来可视化新合成的内源性蛋白质或外源性单分子翻译成像报告，我们发现在动态迁移的细胞中，依赖于APC的RNA的翻译与细胞质位置并不协调，正如目前的范例所表明的那样。相反，我们发现它们的翻译与特定的外周细胞过程相协调，在延长突起/片层脂肪时被激活，在突起回缩时被抑制。此外，沉默与RNA物理状态的变化有关，表现为单个RNA在收回突起的末端聚集成不同种类的颗粒(Moissoglu等人，2019年)。这些团簇让人想起通过液-液分离形成的RNA颗粒。这些发现揭示了动态极化细胞中翻译的一种新的空间调节模式。他们进一步表明，存在协调特定局部细胞行为与相分离的RNA颗粒的组装/拆卸的机制。我们研究了RNA定位对细胞迁移的功能重要性，并取得了重大发现，即特定的蛋白质合成亚细胞位置可以影响编码蛋白质的调节和功能输出。具体地说，以定位的Rab13 RNA为模型，我们已经证明了Rab13 RNA在外围的局部翻译允许新合成的Rab13蛋白与其激活因子RABIF的共翻译结合。这种外围关联是指导Rab13 GTPase活性以促进细胞迁移所必需的，并且主要受Rab13 RNA的位置控制(Moissoglu等人，2020)。这些发现表明，相同的RNA在细胞质的不同位置的翻译可以指导所产生的蛋白质与不同的相互作用网络相关联，并实现不同的功能输出，这意味着总体上对蛋白质的调节。我们的工作提出的前提是，与疾病相关的信号通路可以通过调节RNA翻译的位置在RNA水平上进行调节，而不是在蛋白质上。这一见解为设计潜在的新治疗平台提供了基础。事实上，我们已经利用我们对潜在机制的了解来开发反义寡核苷酸，这种反义寡核苷酸专门干扰特定内源性转录本的定位。使用这种在体外和体内都很容易应用的方法，我们已经证明了不仅在单个细胞的迁移过程中，而且在三维多细胞癌球的集体侵袭过程中，都需要特定的定位RNA。我们现在正在使用癌症侵袭的异种小鼠肿瘤模型来扩展我们的发现(Chrisafis等人，2020)。

The research program focuses on the role of localized RNAs, i.e. RNAs that are targeted to specific subcellular regions, and the consequences of local RNA translation in the processes of cell migration and cancer invasion. This is a markedly understudied area. The research from my group has spearheaded the study of RNAs localized at protrusions of non-neuronal migrating cells. Over the years we have made a number of contributions regarding the underlying molecular mechanisms used to transport RNAs to protrusions and to regulate their translation; the consequences of local RNA translation on protein function; and the relevance of these events to cancer invasion. We have developed methodologies to study protrusion-localized RNAs in 2D and 3D systems, with an emphasis both on the imaging as well as the generation of unbiased methods of image analysis and quantification (Stueland et al., 2019). We have shown that localization of RNAs at protrusions is directed through at least two distinct pathways which depend on different protein factors and are regulated differently by the mechanical properties of the cytoskeleton (Wang et al., 2017). Most of our work has focused on a subset of 70 RNAs which require the tumor suppressor APC for localization. We have shown that localization of APC-dependent RNAs is important for migration in 2D and 3D environments. We have identified cis RNA elements that direct accumulation at protrusions; protein factors that associate with these RNAs and control their transport or translation; and disease mutations that disrupt these processes (Mili et al., 2008; Yasuda et al., 2013; Yasuda et al., 2017; Moissoglu, Pichon et al., in submitted). We have further revealed novel mechanisms controlling localized transcripts, showing that APC-dependent RNAs require detyrosinated microtubules whose formation is promoted by the stiffness of the extracellular matrix, mechanical tension and actomyosin contractility (Wang et al., 2017). We have uncovered a novel mode of translational regulation of localized transcripts. Using state-of-the-art methodologies to visualize newly-synthesized endogenous proteins, or exogenous single-molecule translation imaging reporters, we have discovered that in dynamically migrating cells, translation of APC-dependent RNAs is not coordinated with cytoplasmic position, as the current paradigm suggests. Instead, we found that their translation is coordinated with specific peripheral cellular processes, being activated at extending protrusions/lamellipodia and suppressed upon protrusion retraction. Furthermore, silencing is coupled to a change in the physical state of the RNAs manifested by single RNAs clustering into heterogeneous granules at the tips of retracting protrusions (Moissoglu et al., 2019). These clusters are reminiscent of RNA granules formed by liquid-liquid phase separation. These findings have revealed a novel mode of spatial regulation of translation in dynamically polarized cells. They further suggest the existence of mechanisms that coordinate specific local cellular behaviors with the assembly/disassembly of phase-separated RNA granules. We have investigated the functional importance of RNA localization on cell migration and have made the significant discovery that the particular subcellular site of protein synthesis can affect the regulation and functional output of the encoded protein. Specifically, using the localized RAB13 RNA as a model, we have demonstrated that local translation of the RAB13 RNA at the periphery allows the co-translational association of the newly-synthesized RAB13 protein with its activator, the exchange factor RABIF. This peripheral association is required for directing RAB13 GTPase activity to promote cell migration and is governed primarily by the location of the RAB13 RNA (Moissoglu et al., 2020). These findings revealed that translation of the same RNA in different positions in the cytoplasm can direct the resulting protein to associate with different interacting networks and fulfil different functional outputs, with implications regarding protein regulation in general. The premise set forth by our work is that signaling pathways relevant to disease can be regulated, not at the protein, but rather at the RNA level through modulating the location of RNA translation. This insight offers the basis for the design of potential new therapeutic platforms. Indeed, we have exploited our knowledge of the underlying mechanisms to develop antisense oligonucleotides that interfere specifically with localization of particular endogenous transcripts. Using this methodology, which can be easily applied both in vitro and in vivo, we have demonstrated a requirement for specific localized RNAs not only during migration of individual cells, but also during collective invasion of 3-dimensional multicellular cancer spheroids. We are now extending our findings using xenograft mouse tumor models of cancer invasion (Chrisafis et al., 2020).