Tools for studying the regulation of Homer protein splicing

研究荷马蛋白剪接调节的工具

• 批准号: 10349911
• 负责人: Paul J. Kammermeier
• 金额: $ 15.4万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10349911
• 关键词: Alternative Splicing; Amino Acids; Animal Model; Animals; Binding; C-terminal; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Detection; Disease; Dominant-Negative Mutation; Epilepsy; Exhibits; Future; Genes; Glutamates; Green Fluorescent Proteins; Homer 1; Homer 1a; Homer 3; Homer protein; Investigation; Knock-in; Lead; Length; Literature; Long-Term Depression; Messenger RNA; Metabotropic Glutamate Receptors; Molecular; Monitor; Mus; Nervous System Trauma; Oranges; Outcome; Pathway interactions; Pharmacology; Physiological; Process; Protein Isoforms; Protein Splicing; Proteins; RNA Splicing; Receptor Signaling; Regulation; Resistance; Scaffolding Protein; Seizures; Signal Transduction; Stimulus; Synapses; Testing; Time; Tissues; Transcript; Transgenic Mice; Translations; Trauma; Variant; Wild Type Mouse; cell type; cupidin; density; embryonic stem cell; fluorophore; mRNA Precursor; metabotropic glutamate receptor 7; neuronal excitability; neuroregulation; new therapeutic target; overexpression; postsynaptic; receptor coupling; relating to nervous system; response; scaffold; stem; synaptic inhibition; tool

Abstract Homer scaffolding proteins are important regulators of glutamatergic synapses that organize metabotropic glutamate receptor (mGluRs 1&5) signaling domains near the post-synaptic density (PSD) where they can induce short and long term synaptic inhibition. Neural activity and other stimuli induce a shift from the constitutively expressed, long Homer1b and 1c isoforms to the immediate early (IEG) Homer1a splice variant. Because Homer1a can interact with binding partners but cannot multimerize to promote scaffolding, it disrupts Homer scaffolds and disperses mGluR1 and 5 from the PSD. Thus, Homer1a acts as a natural dominant negative. Indeed, expression Homer1a can uncouple mGluR1 & 5 from postsynaptic processes such as short and long term depression while protecting mGluR coupling to non-synaptic effectors. By elevating Homer1a expression, cells profoundly change mGluR-effector coupling, acting as a molecular switch for mGluR signaling. In this way, regulation of Homer1 splicing is an important means of altering synaptic efficacy. To date however, much remains to be learned about the regulation and expression of Homer1a, including whether Homer1 mRNA splicing and translation occurs locally, near the post-synapse as is the case for many other synaptic proteins. Further, the mechanisms leading to Homer1a splicing are only poorly understood. These questions are relevant not only to the pathophysiological instance in disorders such as epilepsy, but also to the normal physiological situation in which Homer1a is more modestly up, and down regulated in response to more subtle changes in neural firing. To this end, we will generate a mouse using CRISPR in which the endogenous Homer1a protein will be C-terminally tagged with a green fluorescent protein (GFP) and the long isoforms with a red fluorophore (mScarlet-H) so that their expression levels, time course, and sub-cellular distribution can be analyzed following insult and pharmacological manipulation. These questions and others will benefit from the animal model we will generate from this proposal. To achieve this, we will pursue the following Specific Aims: Aim 1: To optimize and test the differentially tagged long and short Homer proteins in mouse embryonic stem cells. Aim 2: To generate a mouse in which the endogenous short and long Homer proteins are differentially tagged with green (GFP) and red (mScarlet-H) florescent proteins.

抽象的 荷马脚手架蛋白是组织代谢型的谷氨酸能突触的重要调节剂 谷氨酸受体（mglurs 1＆5）信号域附近，突触后密度（PSD）可以在其中可以 诱导短期和长期的突触抑制作用。神经活动和其他刺激引起了从 组成型表达的，长的Homer1b和1C同工型，直接提早（IEG）HOMER1A剪接变体。 因为HOMER1A可以与绑定伙伴互动，但不能多次磨合以促进脚手架，所以它破坏了 荷马脚手架和分散pSD的mglur1和5。那就是荷马1a充当自然的主导地位 消极的。实际上，homer1a表达可以从突触后过程（例如短暂的过程）中脱离mglur1＆5 和长期的抑郁症，同时保护与非突触作用的mglur耦合。通过提升Homer1a 表达，细胞深刻地改变了mglur-effter耦合，充当mglur的分子开关 信号。这样，Homer1剪接的调节是改变突触效率的重要手段。迄今为止 但是，关于Homer1a的调节和表达仍有很多待了解，包括是否存在 HOMER1 mRNA剪接和翻译在本地发生，在后散布附近，就像许多其他情况一样 突触蛋白。此外，导致HOMER1A剪接的机制仅被鲜为人知。这些 问题不仅与癫痫等疾病的病理生理实例有关，而且与 Homer1a更适度的正常身体状况，并根据更多 神经结尾的细微变化。为此，我们将使用CRISPR生成鼠标，其中内源性 HOMER1A蛋白将用绿色荧光蛋白（GFP）和长的同工型在C末端标记 红色的浮游植物（Mscarlet-H），以便它们的表达水平，时间过程和细胞亚分布可以是 受伤和药物操纵后进行了分析。这些问题和其他问题将从 我们将从该提案中产生动物模型。为了实现这一目标，我们将追求以下特定目标： 目标1：优化和测试鼠标中不同标记的长长和短本垒打蛋白 胚胎干细胞。目标2：生成内源性短而长本垒的鼠标 蛋白质用绿色（GFP）和红色（MSCARLET-H）植物蛋白标记不同。