Regulation and functional effects of localized RNAs

局部RNA的调控和功能效应

• 批准号: 10702578
• 负责人: Stavroula Mili
• 金额: $ 142.05万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702578
• 关键词: 3-Dimensional; APC gene; Actomyosin; Affect; Antisense Oligonucleotides; Area; Cancer Model; Cell physiology; Cells; Coupled; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; Disease; Disease Progression; Elements; Environment; Epithelial Cells; Event; Extracellular Matrix; Generations; Genetic Translation; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Image; Image Analysis; In Vitro; Individual; Knowledge; Liquid substance; Location; Malignant Neoplasms; Mammalian Cell; Mechanics; Mesenchymal; Messenger RNA; Methodology; Microtubules; Modeling; Molecular; Mus; Mutation; Neoplasm Metastasis; Neurons; Oocytes; Output; Pathway interactions; Peripheral; Phase; Positioning Attribute; Process; Protein Biosynthesis; Proteins; RNA; Regulation; Reporter; Research; Role; Signal Pathway; Site; System; Transcript; Translational Regulation; Translations; Work; Xenograft procedure; cancer cell; cancer type; cell behavior; cell motility; combat; design; image translation; imaging modality; in vivo; insight; interest; mechanical properties; migration; novel; novel therapeutics; physical state; polarized cell; programs; protein function; single molecule; therapeutic target; tumor

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.

该研究计划的重点是局部mRNA的作用，即针对特定亚细胞区域的mRNA，以及局部mRNA翻译对编码蛋白功能的后果。大多数关注这些过程和潜在机制的研究主要是在大型和稳定的极化系统（例如卵母细胞和神经元）中进行的。相反，在细胞迁移和癌症侵袭的背景下，我们正在小型动态细胞（例如间质和上皮细胞）中研究这些事件。这是一个明显研究的区域。具体而言，我小组的研究率先研究了位于非神经迁移细胞突起的mRNA。多年来，我们对用于将mRNA转运到突起并调节其翻译的基本分子机制做出了许多贡献。局部mRNA翻译对蛋白质功能的后果；这些事件与癌症入侵的相关性。 1）我们已经开发了研究2D和3D系统中的突发定位的mRNA的方法，重点是成像以及图像分析和定量的无偏见方法（Stueland等人，2019; Chrisafis等，2020; Moissoglu等人）。 2）我们已经表明，在突起处的mRNA定位是通过至少两种依赖不同蛋白质因子的途径引导的，并通过细胞骨架的机械性能对不同的调节（Wang等，2017; Moriarty等，2022）。我们的大部分工作都集中在70个mRNA的子集上，这些mRNA需要抑制肿瘤APC进行定位。我们已经表明，依赖APC的RNA的定位对于在2D和3D环境中迁移很重要。我们已经确定了直接在突起处积累的顺式RNA元素。与这些mRNA相关并控制其运输或翻译的蛋白质因子；和破坏这些过程的疾病突变（Mili等，2008； Yasuda等，2013； Yasuda等，2017； Moissoglu，Pichon等，2021）。 3）我们进一步揭示了控制局部转录物的新型机制，表明APC依赖性mRNA需要驱虫式的微管，其形成是由细胞外基质的刚度，机械张力和肌动肌球蛋白收缩力促进的（Wang等人，2017年； Moriarty等，2022222）。 4）我们发现了一种新型的局部转录物调节模式。具体而言，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记者，我们发现在动态迁移的细胞中，APC依赖性mRNA的翻译与当前的范式相吻合，并不是与细胞质位置相协调的。取而代之的是，我们发现它们的翻译与特定的外围细胞过程协调，并在扩展突起/薄片脂肪症时被激活，并在撤回突起时被抑制。此外，在缩回突起的尖端，单个mRNA聚集到异质颗粒中表现出的mRNA的物理状态的变化（Moissoglu等，2019）。这些簇让人联想到由液态液相分离形成的RNA颗粒。这些发现揭示了动态极化细胞中平移的空间调节的新型模式。他们进一步表明存在将特定局部细胞行为与相分开的RNA颗粒的组装/拆卸的机制存在。 5）我们已经研究了RNA定位在细胞迁移上的功能重要性，并已经显着发现，蛋白质合成的特定亚细胞位点可以影响编码蛋白的调节和功能输出。具体而言，使用局部化RAB13 RNA作为模型，我们已经证明了在周围的Rab13 mRNA的局部翻译允许新结合的Rab13蛋白与其激活剂Rabif的激活剂Rabif的共同翻译关联。指导RAB13 GTPase活性以促进细胞迁移需要这种外围关联，并且主要受Rab13 mRNA的位置（Moissoglu等，2020）。这些发现表明，在细胞质中不同位置的同一mRNA的翻译可以指导所得蛋白质与不同的相互作用网络相关，并实现不同的功能输出，这对蛋白质调节的含义一般。我们工作提出的前提是，与疾病相关的信号通路可以受到调节，而不是在蛋白质上，而是通过调节mRNA翻译的位置来调节RNA水平。该洞察力为设计潜在的新治疗平台设计提供了基础。的确，我们利用了对基本机制的了解，以开发反义寡核苷酸，这些反义寡核苷酸特异性地干扰了特定内源性转录本的定位。使用这种方法，可以轻松地在体外和体内应用，我们已经证明了对特定局部RNA的要求，不仅在单个细胞的迁移过程中，而且在集体入侵三维多细胞癌球体期间（Chrisafis等人，2020年）。现在，我们使用异种移植小鼠肿瘤模型的癌症侵袭来扩展发现。