The Role of NMD in Olfactory Neurogenesis

描述（由申请人提供）：本提案的重点是高度保守的RNA降解途径-无意义介导的RNA衰变(NMD)-最初被定义为降解异常mrna的质量控制机制，但现在被认为是正常基因表达的重要调节因子。特定的NMD分支已经被定义，每个分支降解正常mrna的不同亚群，导致每个分支调节不同的生物事件的假设。我的实验室专注于NMD途径的一个特定分支，该分支依赖于两个相关的蛋白质- upf3a和upf3b -由一个进化上古老的基因平行对编码，自脊椎动物出现以来就存在。由于人类UPF3B突变导致智力残疾，人们对NMD的UPF3依赖分支非常感兴趣。此外，UPF3A和UPF3B的突变与人类神经发育障碍显著相关。在这项应用中，我们建议通过使用我们最近生成的Upf3a和upf3b突变小鼠模型来破译NMD控制神经系统发育和功能的潜在机制。我们发现UPF3B缺失的小鼠具有模仿人类携带UPF3B突变的行为缺陷。这些突变小鼠在嗅觉神经发生方面也有显著缺陷，这表明NMD对嗅觉感觉神经元（OSN）的存活、成熟和轴突引导至关重要。在本提案中，我们利用嗅觉系统的技术优势和我们的小鼠模型来阐明NMD在生物学和分子水平上控制神经系统发育和功能的潜在机制。在Aim 1中，我们将继续我们的初步研究，表明UPF3B控制两种主要类型的osn的平衡，它们与中枢神经系统（CNS）的连接以及嗅觉行为。在Aim 2中，我们建议使用尖端的方法在体内鉴定UPF3B靶向衰变的mrna -全基因组范围。这一点很重要，因为很少有直接的NMD靶mrna被鉴定出来，而且大多数被鉴定出来的mrna都是在细胞系中发现的。我们将解决的另一个被忽视的研究领域是NMD是否对细胞不同区域的靶mrna进行差异调节。这一点很重要，因为越来越清楚的是，包括神经元在内的许多极化细胞会将事件分区化以提高效率。在Aim 3中，我们将重点关注UPF3A及其与UPF3B的关系。我们之前报道过UPF3A蛋白在UPF3B缺失的情况下显著稳定，这表明后者补偿了前者，这一假设得到了临床证据的支持。我们最近获得的初步证据表明UPF3A也可以作为NMD抑制因子发挥作用。通过比较Upf3a/Upf3b双ko小鼠与Upf3a和Upf3b单ko小鼠以及复合杂合子的嗅觉缺陷和失调转录本，我们提出定义Upf3a和Upf3b在体内独特的、冗余的和拮抗的功能。

DESCRIPTION (provided by applicant): This proposal is focused on a highly conserved RNA degradation pathway-Nonsense-Mediated RNA Decay (NMD)-that was originally defined as a quality control mechanism that degrades aberrant mRNAs, but now is recognized as an important regulator of normal gene expression. Specific NMD branches have been defined, each of which degrades different subsets of normal mRNAs, leading to the hypothesis that each branch regulates distinct biological events. My laboratory is focused on a specific branch of the NMD pathway that depends on two related proteins-UPF3A and UPF3B-encoded by an evolutionarily ancient gene paralog pair that has existed since the emergence of vertebrates. There is considerable interest in the UPF3- dependent branch of NMD because mutations in UPF3B in humans cause intellectual disability. Furthermore, mutations in both UPF3A and UPF3B are significantly associated with neuro-developmental disorders in humans. In this application, we propose to decipher the underlying mechanisms by which NMD controls nervous system development and function by using Upf3a- and Upf3b-mutant mouse models we recently generated. We find that Upf3b-null mice have behavioral defects that mimic some of those in humans harboring UPF3B mutations. These mutant mice also have striking defects in olfactory neurogenesis that suggest that NMD is critical for olfactory sensory neuron (OSN) survival, maturation, and axon guidance. In this proposal, we leverage the technical advantages of the olfactory system and our mouse models to elucidate the underlying mechanisms by which NMD controls nervous system development and function at both the biological and molecular levels. In Aim 1, we will follow-up on our preliminary studies suggesting that UPF3B controls the balance of the two major types of OSNs, their connections with the central nervous system (CNS), and olfactory behavior. In Aim 2, we propose to use cutting-edge methods to identify-genome-wide-the mRNAs targeted for decay by UPF3B in vivo. This is important, since very few direct NMD target mRNAs have been identified, and most of those that have been identified were discovered in cell lines. Another neglected area of investigation that we will address is whether NMD differentially regulates target mRNAs in different regions of a cell. This is important since it is becoming increasingly clear that many polarized cells, including neurons, sub-compartmentalize events to increase efficiency. In Aim 3, we focus on UPF3A and its relationship with UPF3B. We previously reported that UPF3A protein is dramatically stabilized in response to loss of UPF3B, which suggests that the latter compensates for the former, a postulate that is supported by clinical evidence. We recently obtained preliminary evidence that UPF3A can also function as a NMD repressor. By comparing both the olfactory defects and mis-regulated transcripts in Upf3a/Upf3b double-KO mice with those of Upf3a and Upf3b single-KO mice, as well as compound heterozygotes, we propose to define unique, redundant, and antagonistic functions of UPF3A and UPF3B in vivo.