The Role of NMD in Olfactory Neurogenesis

描述(申请人提供)：这项提案集中在一种高度保守的RNA降解途径--无意义介导的RNA衰退(NMD)--最初被定义为一种降解异常mRNAs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。已经定义了特定的NMD分支，每个分支降解正常mRNAs的不同子集，导致假设每个分支调节不同的生物事件。我的实验室专注于NMD途径的一个特定分支，它依赖于两个相关的蛋白质-UPF3A和UPF3B-由一对进化上古老的基因副对编码，自脊椎动物出现以来就一直存在。对依赖UPF3的NMD分支有相当大的兴趣，因为UPF3B在人类中的突变会导致智力残疾。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建立的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缺失的小鼠有一些行为缺陷，与携带UPF3B突变的人类的行为缺陷相似。这些突变的小鼠在嗅神经发生方面也有显著的缺陷，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。在这个方案中，我们利用嗅觉系统和我们的小鼠模型的技术优势来阐明NMD在生物和分子水平上控制神经系统发育和功能的潜在机制。在目标1中，我们将继续我们的初步研究，表明UPF3B控制着两种主要类型的OSNs的平衡，它们与中枢神经系统(CNS)的联系，以及嗅觉行为。在目标2中，我们建议使用尖端方法来识别-基因组范围-UPF3B在体内针对衰退的mRNAs。这一点很重要，因为很少有直接针对NMD的mRNAs被识别出来，而大多数已经被识别的mRNAs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是否以不同的方式调节细胞不同区域的靶向mRNAs。这一点很重要，因为越来越明显的是，许多极化细胞，包括神经元，将事件细分以提高效率。在目标3中，我们重点介绍了UPF3A及其与UPF3B的关系。我们以前报道过，UPF3A蛋白在UPF3B丢失后显著稳定，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错误调节转录本，我们建议确定UPF3A和UPF3B在体内的独特、冗余和拮抗功能。

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.