The Role of NMD in Olfactory Neurogenesis

描述（由申请人提供）：该提案集中于高度保守的RNA降解途径-无义介导的RNA衰变（NMD）-其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。已经定义了特定的NMD分支，每个分支降解正常mRNA的不同子集，导致每个分支调节不同的生物学事件的假设。我的实验室专注于NMD通路的一个特定分支，该通路依赖于两种相关蛋白-UPF 3A和UPF 3B-由一对进化上古老的基因paramount编码，该基因paramount自脊椎动物出现以来就存在。对NMD的UPF 3依赖性分支有相当大的兴趣，因为人类UPF 3 B的突变导致智力残疾。此外，UPF 3A和UPF 3B的突变与人类神经发育障碍显著相关。在本申请中，我们建议使用我们最近生成的Upf 3a和Upf 3b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，Upf 3b基因缺失的小鼠具有行为缺陷，这些行为缺陷模仿了人类中携带UPF 3B突变的一些行为缺陷。这些突变小鼠在嗅觉神经发生方面也有明显的缺陷，这表明NMD对嗅觉感觉神经元（OSN）的存活、成熟和轴突导向至关重要。在这个提议中，我们利用嗅觉系统和我们的小鼠模型的技术优势，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，我们将跟进我们的初步研究，这些研究表明UPF 3B控制两种主要类型的OSN的平衡，它们与中枢神经系统（CNS）和嗅觉行为的联系。在目标2中，我们建议使用尖端的方法来识别基因组范围内的靶向衰变的UPF 3B在体内的mRNA。这一点很重要，因为很少有直接的NMD靶mRNA被鉴定出来，而且大多数已经鉴定出来的mRNA都是在细胞系中发现的。另一个被忽视的研究领域，我们将解决的是是否NMD差异调节靶mRNA在不同区域的细胞。这一点很重要，因为越来越清楚的是，许多极化细胞，包括神经元，亚区室化事件以提高效率。在目标3中，我们关注UPF 3A及其与UPF 3B的关系。我们以前报道过，UPF 3A蛋白在UPF 3B丢失时显着稳定，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，UPF 3A也可以作为一个NMD阻遏物。通过比较Upf 3a/Upf 3b双基因敲除小鼠与Upf 3a和Upf 3b单基因敲除小鼠以及复合杂合子的嗅觉缺陷和误调节转录本，我们提出了在体内定义UPF 3A和UPF 3B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.