Regulation and functional effects of localized RNAs

该研究计划的重点是局部RNA的作用，即靶向特定亚细胞区域的RNA，以及局部RNA翻译在细胞迁移和癌症侵袭过程中的后果。这是一个明显未被充分研究的领域。我的团队的研究率先研究了位于非神经元迁移细胞突起处的RNA。多年来，我们已经作出了一些贡献，关于用于运输RNA突起和调节其翻译的潜在分子机制;局部RNA翻译对蛋白质功能的后果;以及这些事件与癌症侵袭的相关性。我们已经开发了在2D和3D系统中研究弥散定位的RNA的方法，重点是成像以及图像分析和定量的无偏方法的产生（Stueland等人，2019年）。我们已经表明，RNA在突起处的定位是通过至少两种不同的途径来指导的，这两种途径取决于不同的蛋白质因子，并且受到细胞骨架的机械性质的不同调节（Wang等人，2017年）。我们的大部分工作集中在一个子集的70 RNA，需要肿瘤抑制APC的定位。我们已经表明，APC依赖性RNA的定位对于2D和3D环境中的迁移是重要的。我们已经鉴定了指导在突起处积累的顺式RNA元件;与这些RNA相关并控制其转运或翻译的蛋白质因子;以及破坏这些过程的疾病突变（Mili等人，2008; Yasuda等人，2013; Yasuda等人，2017; Moissoglu，Pichon等人，已提交）。我们进一步揭示了控制局部转录物的新机制，表明APC依赖性RNA需要脱酪氨酸的微管，其形成由细胞外基质的硬度、机械张力和肌动球蛋白收缩性促进（Wang et al.，2017年）。我们已经发现了一种新的本地化转录本的翻译调控模式。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分子，我们发现在动态迁移的细胞中，APC依赖性RNA的翻译与细胞质位置不协调，正如目前的范例所示。相反，我们发现它们的翻译与特定的外周细胞过程相协调，在延伸突起/板状伪足处被激活，并在突起缩回时被抑制。此外，沉默与RNA的物理状态的变化相关联，所述变化表现为单个RNA在缩回突起的尖端聚集成异质颗粒（Moissoglu等人，2019年）。这些簇使人联想到通过液-液相分离形成的RNA颗粒。这些发现揭示了动态极化细胞中翻译的空间调节的新模式。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/分解的机制。我们已经研究了RNA定位在细胞迁移中的功能重要性，并且已经发现蛋白质合成的特定亚细胞位点可以影响编码蛋白质的调节和功能输出。具体而言，使用本地化的RAB 13 RNA作为模型，我们已经证明，在周边的RAB 13 RNA的本地翻译允许新合成的RAB 13蛋白与其激活剂，交换因子RABIF的共翻译协会。这种外周结合是指导RAB 13 GT3活性以促进细胞迁移所必需的，并且主要由RAB 13 RNA的位置控制（Moissoglu等人，2020年）。这些发现表明，相同RNA在细胞质中不同位置的翻译可以指导所得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，这对蛋白质调控具有普遍意义。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翻译的位置而不是在蛋白质水平上进行调节。这种见解为设计潜在的新治疗平台提供了基础。事实上，我们已经利用我们的基本机制的知识，开发特异性干扰特定内源性转录本定位的反义寡核苷酸。使用这种方法，它可以很容易地应用于体外和体内，我们已经证明了特定的本地化RNA的要求，不仅在个别细胞的迁移，而且在集体入侵的三维多细胞癌球体。我们现在正在使用癌症侵袭的异种移植小鼠肿瘤模型来扩展我们的发现（Chrisafis等人，2020年）。

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).