SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION IN EPIDERMAL HOMEOSTASIS

小分子非编码RNA对表皮动态平衡中Ras-GTP酶功能的调节 项目摘要/摘要 Ras-MAPK信号转导通路是表皮的重要调节因子，是Ras-MAPK信号转导通路的重要组成部分。 MAPK信号通路抑制表皮分化，是肿瘤发生的主要驱动力。我们的最新发现 SnoRNAs直接与RAS功能相互作用并调节RAS功能代表着我们的 了解小分子GTP酶的调控。使用我们的新型UV-C交联和免疫沉淀 平台，irCLIP，为了表征转录组广泛的RAS超家族GTP酶与RNA的相互作用，我们有 发现了一个丰富而复杂的snoRNA-Ras-GTPase相互作用网络，这表明snoRNAs可能 调节RAS超家族控制下的所有生物过程，包括生化信号转导节点， 肌动蛋白/膜组织、囊泡和细胞内蛋白运输以及核/细胞质运输。 此K01应用程序的长期目标是深入描述监管功能和 小核泡RNA调控RAS及其超家族GTP酶的作用机制 表皮动态平衡。 在目标I中，我们将重点定义C/D盒snoRNA调控RAS的特异性和广度- 超家族GTP酶的功能。我们的初步irCLIP-seq数据显示，所有5个RAS亚家族的成员， RAS、RHO、ARF、RAB和RAN与SNORD50A/B直接相互作用。因此，SNORD50A/B可能是全局的 Ras超家族GTP酶的抑制子已被描述为K-RAS。使用CRISPR/Cas9基因编辑， SNORD50A/B功能丧失研究将测试9个RAS超家族GTP酶的RAS-GTP酶激活水平 横跨所有5个子家族。活性-RAS-GTP酶下游生化通路的激活状态将 如果适用，也可以用IP-激酶分析和/或磷酸化免疫印迹法进行监测。我们的irCLIP-seq数据 还发现RAS亚型与&gt；20C/D盒snoRNAs相互作用，其中几个在 癌症。这支持多个snoRNAs参与RAS功能调控的假说。在AIM IB，我们将使用CRISPR介导的基因编辑来切除原始人中与RAS相互作用的snoRNAs 通过分析Ras-GTP水平、ERK1/2和AKT磷酸化来评估角质形成细胞的功能丧失 水平，以及3D人体组织模型中的表皮动态平衡。总而言之，这一目标将揭示 哪些C/D盒snoRNAs调节RAS和RAS超家族GTP酶的功能。 AIM II旨在研究依赖RNA的RAS蛋白相互作用组的功能。因为. 他们抑制RAS与法尼基转移酶相互作用的能力，我们假设SNORD50A/B 作为适配器来调节特定的RAS-蛋白质相互作用。使用串联亲和纯化和 近端蛋白生物素化(BioID)方法，我们比较了WT与突变体RAS的相互作用 SNORD50A/B/或-/-背景中的WT RAS。这导致了一份精炼的蛋白质相互作用列表，这些相互作用 以SNORD50A/B特有的方式改变，并支持SNORD50A/B调节囊泡的假设 RAS从高尔基体到质膜的运输。CRISPR介导的功能丧失研究将 用于评估这些候选因子如何有助于调节RAS来控制表皮内环境的稳定 在3D组织模型中。以与AIM IIA正交和互补的方式，我将鉴定全球RNA- AIM IIB中依赖的RAS蛋白相互作用。IrCLIP-Adaptor连接的RAS-RNA的SDS-PAGE分析 复合体支持这样的假设，即更多的蛋白质正在与RAS在依赖于RNA的 举止。目的IIB将使用我们的新型irCLIP-MS方法来鉴定这些因素。加在一起， 这一目标将有助于从功能上描述和扩大我们对RNA如何组织RAS的理解 控制表皮动态平衡的蛋白质复合体。

SMALL NON-CODING RNA REGULATION OF RAS-GTPase FUNCTION in EPIDERMAL HOMEOSTASIS PROJECT SUMMARY/ABSTRACT The Ras-MAPK signal transduction pathway is a critical regulator of the epidermis as dysregulation of Ras- MAPK signaling inhibits epidermal differentiation and is a major driver of tumorigenesis. Our recent discovery that snoRNAs directly interact with and regulate Ras function represents a major paradigm shift in our understanding of small GTPase regulation. Using our novel UV-C cross-linking and immunoprecipitation platform, irCLIP, to characterize transcriptome wide RAS-superfamily GTPase interactions with RNA, we have discovered a rich and complex web of snoRNA-RAS-GTPase interactions suggesting that snoRNAs may regulate all biological processes under RAS-superfamily control, including biochemical signaling nodes, actin/membrane organization, vesicular and intracellular protein trafficking and nuclear/cytoplasmic transport. The long term goals of this K01 application are to deeply characterize the regulatory functions and mechanisms of action of small nucelolar RNAs in modulation of Ras and RAS-superfamily GTPases in control of epidermal homeostasis. In Aim I, we will focus on defining the specificity and breadth of C/D box snoRNA modulation of RAS- superfamily GTPase functions. Our preliminary irCLIP-seq data showed that members of all 5 RAS-subfamilies, RAS, RHO, ARF, RAB and RAN, directly interacted with SNORD50A/B. Thus SNORD50A/B may be a global repressor of RAS-superfamily GTPases as has been described for K-Ras. Using CRISPR/Cas9 gene editing, SNORD50A/B loss-of-function studies will test RAS-GTPase activation levels of 9 RAS-superfamily GTPases spanning all 5 subfamilies. Activation status of biochemical pathways downstream of active-RAS-GTPases will also be monitored with IP-kinase assays and/or phospho-immunoblots when applicable. Our irCLIP-seq data also revealed that Ras isoforms interacted with >20 C/D box snoRNAs, several of which are amplified in cancer. This supports the hypothesis that multiple snoRNAs participate in the regulation of Ras function. In Aim IB, we will use CRISPR-mediated gene editing to excise select Ras-interacting snoRNAs from primary human keratinocytes and assess loss-of-function via analysis of Ras-GTP levels, ERK1/2 and AKT phosphorylation levels, and on epidermal homeostasis in 3D human tissue models. Together, this aim will reveal the extent to which C/D box snoRNAs regulate Ras and RAS-superfamily GTPase functions. Aim II is designed to functionally characterize the RNA-dependent Ras protein interactome. Because of their ability to suppress interaction of Ras with farnesyltransferase, we hypothesized that SNORD50A/B function as adaptors to modulate specific Ras-protein interactions. Using a tandem affinity purification and proximal protein biotinylation (BioID) approach, we compared the interactomes of WT to mutant Ras against WT Ras in a SNORD50A/B +/+ or -/- background. This led to a distilled list of protein interactions that were altered in a SNORD50A/B-specific manner and support the hypothesis that SNORD50A/B regulate vesicular trafficking of Ras from the Golgi to the plasma membrane. CRISPR-mediated loss-of-function studies will be used to assess how these candidate factors contribute to regulation of Ras in control of epidermal homeostasis in 3D tissue models. In a manner orthogonal and complementary to Aim IIA, I will identify global RNA- dependent Ras protein interactions in Aim IIB. SDS-PAGE resolution of irCLIP-adaptor ligated Ras-RNA complexes support the hypothesis that additional proteins are being co-purified with Ras in an RNA-dependent manner. Aim IIB will use our novel irCLIP-mass-spectrometry method to identify these factors. Taken together, this aim will serve to functionally characterize and broaden our understanding of how RNA organizes Ras protein complexes to control epidermal homeostasis.