Understanding the regulatory language of RNA localization

摘要 真核细胞包含无数个空间上不同的位置，这些位置服务于各种功能。 为了促进这一组织，真核基因的表达通常通过贩运在空间上进行调控 以及将数千种不同的RNA分子隔离到不同的细胞位置。对此的不当监管 从果蝇的发育缺陷来看，过程导致了一系列系统中的有害表型 人类的神经系统疾病。 尽管这一点很重要，但我们对RNA本地化的调控知识相当有限。对于其他 转录后调控的模式，如剪接，我们对RNA结合相互作用的理解 蛋白质(RBPs)和RNA基序导致的特定结果要成熟得多。这有赖于多年的 许多小组的工作已经定义了剪接的监管语言，并允许我们做出预测性的 以及关于剪接如何在不同条件和细胞环境中受到调控的组合模型。我们 在RNA本地化方面缺乏这样的能力，很大程度上是因为我们缺乏类似的“部件列表” 这定义了本地化监管的措辞。 一般来说，RBP/RNA结合对转录后调控过程的影响，如剪接或 RNA的衰变在不同类型的细胞中是一致的。例如，如果RBP促进了一个细胞内外显子的剪接 在另一种细胞类型中，它通常对该外显子产生类似的影响。然而，由于RNA本地化是 由于固有地与细胞形态有关，跨细胞类型的定位调节的共性尚不清楚。 导致RNA定位到神经元投射的RNA基序和限制性商业惯例的组合也是 广泛存在于非神经细胞类型中。这些RNA是在非神经细胞中交易的吗？如果是，去哪里？ 这些问题的答案首先需要更好地了解基本的监管语言 本地化。这里提出的实验是我们努力定义这种语言和 测试它的通用性。我们已经开发出分离和分析亚细胞转录的方法 神经元和上皮细胞的顶端和基底区的投射。我们将使用这些技术来 一种生化和转录组范围的方法来定义调节定位的RBP/RNA相互作用 哺乳动物的两种细胞类型：神经元和肠上皮细胞。通过识别错误本地化的记录 在RBP缺失的细胞中，我们将确定RBP/RNA的功能相互作用。使用大规模平行的报告化验， 我们将采取一种公正的方法来寻找调节本地化的RNA序列。通过比较 已识别的跨细胞类型的功能性RBP/RNA相互作用的活动，我们将首次能够 直接评估RNA定位的一般性。这种有条不紊和创新的方法是 我们努力阐明这一基本但鲜为人知的细胞过程。

SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous “parts list” that defines the language of localization regulation. Generally, the effect of RBP/RNA binding on post-transcriptional regulatory processes like splicing or RNA decay is consistent across cell types. For example, if an RBP promotes the splicing of an exon in one cell type, it often exerts a similar effect on that exon in another cell type. However, because RNA localization is inherently tied to cell morphology, the generality of localization regulation across cell types is unknown. Combinations of RNA motifs and RBPs that result in RNA localization to projections in neurons are also broadly present in non-neuronal cell types. Are these RNAs trafficked in non-neuronal cells? If so, to where? The answers to these questions first require a better knowledge of the underlying regulatory language of localization. The experiments proposed here are the beginnings of our efforts to define this language and test its generality. We have developed methods to isolate and profile subcellular transcriptomes from the projections of neurons and the apical and basal regions of epithelial cells. We will use these techniques to take a biochemical and transcriptome-wide approach to defining RBP/RNA interactions that regulate localization in two mammalian cell types: neurons and intestinal epithelial cells. By identifying transcripts that are mislocalized in RBP-null cells, we will identify functional RBP/RNA interactions. Using a massively parallel reporter assay, we will take an unbiased approach to finding RNA sequences that regulate localization. By comparing the activities of identified functional RBP/RNA interactions across cell types, we will for the first time be able to directly assess the generality of RNA localization. This methodical and innovative approach is the first step in our efforts to shed light on this fundamental but poorly understood cellular process.