Regulation and functional effects of localized RNAs

该研究项目侧重于局部mRNA的作用，即靶向特定亚细胞区域的mRNA，以及局部mRNA翻译对编码蛋白功能的影响。大多数关于这些过程及其潜在机制的研究大多是在大型和稳定极化系统中进行的，例如卵母细胞和神经元。相反，我们在细胞迁移和癌症侵袭的背景下，在小的、动态的细胞中研究这些事件，如间充质细胞和上皮细胞。这是一个明显未被充分研究的领域。具体来说，我们小组的研究率先研究了定位于非神经元迁移细胞突起的mrna。多年来，我们对用于将mrna转运到突起并调节其翻译的潜在分子机制做出了许多贡献；局部mRNA翻译对蛋白质功能的影响；以及这些事件与癌症侵袭的关系。1)我们开发了在2D和3D系统中研究突出定位mrna的方法，重点是成像以及生成无偏倚的图像分析和量化方法（Stueland等人，2019；Chrisafis等人，2020；Moissoglu等人，出版中）。2)我们已经证明，mrna在突起处的定位是通过至少两种不同的途径进行的，这些途径依赖于不同的蛋白质因子，并受到细胞骨架机械特性的不同调节（Wang等人，2017；Moriarty等人，2022）。我们的大部分工作都集中在70个mrna的子集上，这些mrna需要肿瘤抑制因子APC进行定位。我们已经证明，apc依赖性rna的定位对2D和3D环境中的迁移很重要。我们已经确定了顺式RNA元件，直接积累在突起；与这些mrna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，2021）。3)我们进一步揭示了控制局部转录本的新机制，表明apc依赖性mrna需要去酪化微管，其形成由细胞外基质的刚度、机械张力和肌动球蛋白收缩性促进（Wang et al., 2017; Moriarty et al., 2022）。4)我们发现了一种新的本地化转录本的翻译调控模式。具体来说，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，apc依赖性mrna的翻译与细胞质位置不协调，正如目前的范式所表明的那样。相反，我们发现它们的翻译与特定的外周细胞过程协调，在延伸突起/板足时被激活，在突起收缩时被抑制。此外，沉默与mrna物理状态的变化有关，表现为单个mrna在收缩突起的尖端聚集成异质颗粒（Moissoglu et al., 2019）。这些团簇让人想起液-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一种新的空间调节模式。他们进一步提出存在协调特定局部细胞行为与相分离RNA颗粒组装/拆卸的机制。5)我们研究了RNA定位对细胞迁移的功能重要性，并有重大发现，蛋白质合成的特定亚细胞位点可以影响编码蛋白的调控和功能输出。具体来说，我们以定位的RAB13 RNA为模型，证明了RAB13 mRNA在外周的局部翻译允许新合成的RAB13蛋白与其激活剂交换因子RABIF的共翻译关联。这种外周关联是指导RAB13 GTPase活性以促进细胞迁移所必需的，并且主要由RAB13 mRNA的位置控制（Moissoglu et al., 2020）。这些发现表明，细胞质中不同位置的相同mRNA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，这对一般的蛋白质调节具有重要意义。我们的工作提出的前提是，与疾病相关的信号通路可以被调节，不是在蛋白质上，而是在RNA水平上，通过调节mRNA翻译的位置。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，这些反义寡核苷酸特异性地干扰特定内源性转录本的定位。使用这种可以很容易地在体外和体内应用的方法，我们已经证明，不仅在单个细胞的迁移过程中，而且在三维多细胞癌球体的集体侵袭过程中，都需要特定的定位rna （Chrisafis等人，2020）。我们现在正在扩展我们的发现，使用异种移植小鼠肿瘤模型的癌症侵袭。

The research program focuses on the role of localized mRNAs, i.e. mRNAs that are targeted to specific subcellular regions, and on the consequences that local mRNA translation has on the function of the encoded proteins. Most studies focusing on these processes and the underlying mechanisms have been carried out mostly in large and stably polarized systems, such as oocytes and neurons. We are instead studying these events in small, dynamic cells such as mesenchymal and epithelial cells, in the context of cell migration and cancer invasion. This is a markedly understudied area. Specifically, the research from my group has spearheaded the study of mRNAs 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 mRNAs to protrusions and to regulate their translation; the consequences of local mRNA translation on protein function; and the relevance of these events to cancer invasion. 1) We have developed methodologies to study protrusion-localized mRNAs 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; Chrisafis et al., 2020; Moissoglu et al., in press). 2) We have shown that localization of mRNAs 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; Moriarty et al., 2022). Most of our work has focused on a subset of 70 mRNAs 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 mRNAs 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., 2021). 3) We have further revealed novel mechanisms controlling localized transcripts, showing that APC-dependent mRNAs require detyrosinated microtubules whose formation is promoted by the stiffness of the extracellular matrix, mechanical tension and actomyosin contractility (Wang et al., 2017; Moriarty et al., 2022). 4) We have uncovered a novel mode of translational regulation of localized transcripts. Specifically, 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 mRNAs 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 mRNAs manifested by single mRNAs 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. 5) 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 mRNA 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 mRNA (Moissoglu et al., 2020). These findings revealed that translation of the same mRNA 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 mRNA 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 (Chrisafis et al., 2020). We are now extending our findings using xenograft mouse tumor models of cancer invasion.