Tools for studying the regulation of Homer protein splicing

摘要 荷马支架蛋白是组织代谢性谷氨酸能突触的重要调节因子 谷氨酸受体(mGluRs 1和5)信号域靠近突触后密度(PSD)的地方 诱导短期和长期的突触抑制。神经活动和其他刺激会导致从 结构性表达，长Hmer 1b和1c亚型为即刻早期(IEG)Hmer 1a剪接变异体。 由于Hmer 1a可以与绑定伙伴交互，但不能通过多聚化来促进脚手架，因此它破坏了 荷马支架并将mGluR1和5从PSD中分散出来。因此，Hmer 1a扮演着自然显性的角色 不是。事实上，表达Hmer 1a可以将mGluR1和5从突触后过程中解脱出来，例如Short 长期抑郁，同时保护mGluR与非突触效应器的偶联。通过提升Hmer 1a 表达，细胞深刻改变mGluR-效应器偶联，成为mGluR的分子开关 发信号。在这种情况下，Hmer-1剪接的调节是改变突触EF-fi敏感性的重要手段。到目前为止 然而，关于Hmer 1a的调控和表达仍有许多需要了解的地方，包括是否 Hmer 1mRNA的剪接和翻译发生在突触后附近的局部，就像许多其他 突触蛋白。此外，导致Hmer 1a剪接的机制还知之甚少。这些 问题不仅与癫痫等疾病的病理生理学情况有关，而且与 正常的生理状态，在这种情况下，Hmer 1a相对较温和地上调和下调，以响应更多 神经fi环的细微变化。为此，我们将利用CRISPR产生一只小鼠，在其中内源性 Hom1a蛋白的C末端将标记有绿色fl荧光蛋白(Gfp)，长的异构体将带有 红色fl载体(mScarlet-H)，以使其表达水平、时间进程和亚细胞分布 分析了侮辱和药物操纵后的情况。这些问题和其他问题将使fit受益于 我们将从这个提案中生成动物模型。为了实现这一目标，我们将实现以下具体目标：fic： 目的1：优化和检测差异标记的长、短Hmer蛋白在小鼠体内的表达 胚胎干细胞。目的2：创造一种内源性短荷马和长荷马 蛋白质分别用绿色(Gfp)和红色(mScarlet-H)fl荧光蛋白标记。

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.