Genome-wide identification of mRNA localization motifs and factors

描述（由申请人提供）：基因表达的控制在许多阶段受到调节。这种调控的一个方面，即特定mRNA转录物在不同细胞位置的定位，直到最近才得到充分的认识。这些信息的正确定位对于许多细胞和高阶过程至关重要。例如，在小鼠中，树突靶向特定信息的破坏导致长期增强的减少，这意味着这一过程可能对学习和记忆很重要。最近的研究表明，在神经元细胞的轴突上有多达2000个mrna。然而，关于消息如何被标记为传输或中介它的因素所知甚少。虽然少数信息的定位机制已经被解剖，但这些机制如何在转录组范围内运作尚不清楚。本提案概述的研究旨在定义负责许多信息定位的顺式作用序列和反式作用因子。具体来说，我将结合RNA生物化学、分子遗传学和计算基因组学来解决这个问题。使用来自轴突和细胞体的RNA-seq，我将定义定位特定信息所需的RNA序列。然后，我将使用MS2-GFP报告系统来完善这些序列，并通过计算和实验来搜索这些基序的其他实例，无论它们是否包含在初级或次级RNA结构中。结合这些基序的因素将被生物化学鉴定，它们对其他信息定位的影响也将被鉴定

DESCRIPTION (provided by applicant): Control of gene expression is regulated at many stages. One aspect of this regulation, the localization of specific mRNA transcripts to distinct cellular locations, has only recently been fully appreciated. Correct targeting of these messages can be crucial for many cellular and higher-order processes. For example, disruption of dendritic targeting of specific messages in mice leads to a decrease in long-term potentiation, implying that this process may be important for learning and memory. Recent studies have shown that up to two thousand mRNAs are localized to axons in neuronal cells. However, little is known about how messages are tagged for transport or the factors that mediate it. Although the mechanism of localization of a handful of messages has been dissected, how such mechanisms may operate on a transcriptome-wide scale is unknown. The studies outlined in this proposal aim to define the cis-acting sequences and trans-acting factors responsible for the localization of many messages. Specifically, I will address this question using a combination of RNA biochemistry, molecular genetics, and computational genomics. Using RNA-seq from axons and cell bodies, I will define the RNA sequences necessary for localization of specific messages. I will then refine these sequences using an MS2-GFP reporter system and search the localized transcriptome both computationally and experimentally for other instances of these motifs, whether they be contained in primary or secondary RNA structure. The factors that bind these motifs will then be identified biochemically and their effects on the localization of other messages will be identified using RNAi and localized RNA-seq. The results of these studies will allow the identification of families of messages that use similar motifs and factors for their localization, as well as the prediction of motif sequences in other messages. This has important clinical and therapeutic implications as mutations in many RNA-localizing proteins are associated with several neuronal diseases, including spinomuscular atrophy and ALS.