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

小非编码 RNA 对表皮稳态中 RAS-GTP 酶功能的调节

• 批准号: 9293751
• 负责人: Brian J Zarnegar
• 金额: $ 12.74万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9293751
• 关键词: Actins; Advanced Development; Affinity Chromatography; Biochemical; Biochemical Pathway; Biological Assay; Biological Process; Biotinylation; CRISPR/Cas technology; Cell Proliferation; Cell membrane; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Disease; Endocytic Vesicle; Epidermal Growth Factor Receptor; Epidermis; Epithelial; Equilibrium; Family; GTPase-Activating Proteins; Gene Expression; Genes; Genetic; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Homeostasis; Human; Hybrids; Immunoprecipitation; Internet; KRAS2 gene; MAP Kinase Gene; MAPK3 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Methods; Modeling; Molecular Conformation; Monitor; Monomeric GTP-Binding Proteins; Mutation; Nature; Nuclear; Oncogenic; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Protein Isoforms; Protein Microchips; Proteins; Proto-Oncogene Proteins c-akt; Psoriasis; RNA; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Resolution; Role; Signal Transduction; Signal Transduction Pathway; Skin; Small Nucleolar RNA; Small RNA; Specificity; Stem cells; Testing; Tissue Model; Tissues; Untranslated RNA; Work; chronic wound; crosslink; design; human tissue; in vivo; keratinocyte; loss of function; member; mutant; novel; prevent; protein complex; protein protein interaction; protein transport; ras Proteins; rho; self-renewal; trafficking; transcriptome; tumorigenesis

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.

表皮稳态中RAS-GTPase功能的小非编码RNA调节 项目摘要/摘要 RAS-MAPK信号转导途径是表皮的关键调节剂，因为Ras-的失调失调 MAPK信号传导抑制表皮分化，是肿瘤发生的主要驱动力。我们最近的发现 Snornas直接与RAS函数直接相互作用，代表了我们的主要范式转变 了解小型GTPase调节。使用我们新颖的UV-C交联和免疫沉淀 平台，IRCLIP，以表征转录组宽Ras-Superfamily GTPase与RNA的相互作用，我们有 发现了一个丰富而复杂的snorna-ras-gtpase相互作用的网络，这表明snornas可能 调节Ras-Superflamily控制下的所有生物过程，包括生化信号节点， 肌动蛋白/膜组织，囊泡和细胞内蛋白运输以及核/细胞质转运。 该K01应用程序的长期目标是深层表征监管功能和 小核RNA在对照中的RAS和RAS-Superfamily GTPase调制中的小核RNA的作用机制 表皮稳态。 在AIM I中，我们将专注于定义C/D盒子的特异性和广度。 超家族GTPase函数。我们的初步IRCLIP-seq数据显示，所有5个RAS-育家庭的成员， Ras，Rho，Arf，Rab和Ran直接与Snord50a/b进行了交互。因此，snord50a/b可能是全球 如K-Ras所述，Ras-Superfamily GTPases的阻遏物。使用CRISPR/CAS9基因编辑， SNORD50A/B功能丧失研究将测试9 Ras-Superfamily GTPase的Ras-GTPase激活水平 跨越所有5个亚家族。 Active-Ras-GTPases下游的生化途径的激活状态将 如果适用，也可以使用IP-激酶测定法和/或磷酸化免疫印迹来监测。我们的IRCLIP-SEQ数据 还揭示了RAS同工型与> 20 c/d盒snornas相互作用，其中一些被放大 癌症。这支持了以下假设：多个snornas参与RAS功能的调节。目标 IB，我们将使用CRISPR介导的基因编辑来消费税从主要人 角质形成细胞和通过分析Ras-GTP水平ERK1/2和AKT磷酸化来评估功能丧失 水平，以及3D人体组织模型中的表皮稳态。这个目标在一起将揭示 哪个C/D盒snornas调节RAS和RAS-Superfamily GTPase功能。 AIM II旨在在功能上表征RNA依赖性RAS蛋白相互作用组。由于 它们抑制RAS与Farnesylsylansferase相互作用的能力，我们假设SNORD50A/B 充当调节特定RAS-蛋白质相互作用的适配器。使用串联亲和力净化和 在 wt ras在snord50a/b +/ +或 - / - 背景中。这导致了蒸馏的蛋白质相互作用清单 以SNORD50A/B特异性方式改变，并支持SNORD50A/B调节水泡的假设 将拉斯从高尔基体贩运到质膜。 CRISPR介导的功能丧失研究将是 用于评估这些候选因素如何导致对表皮稳态控制RA的调节 在3D组织模型中。以某种方式正交和互补的目标IIA，我将确定全球RNA- AIM IIB中的依赖性RAS蛋白相互作用。 SDS-PAGE分辨率的IRCLIP-AUPATTOR连接RAS-RNA 复合物支持以下假设：在RNA依赖性中，其他蛋白质与RAS共纯化 方式。 AIM IIB将使用我们新颖的IRCLIP质量 - 谱法来识别这些因素。在一起， 该目标将在功能上表征和扩大我们对RNA如何组织RA的理解 蛋白质复合物控制表皮稳态。