Cellular RNA binding and regulation by NFX1-123 and its perturbation by high risk human papillomavirus E6

NFX1-123 的细胞 RNA 结合和调节及其受高危人乳头瘤病毒 E6 的干扰

• 批准号: 9597702
• 负责人: Rachel Adria Katzenellenbogen
• 金额: $ 2.85万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9597702
• 关键词: Adult; Affect; Amino Acid Motifs; Antibodies; Binding; Biological Models; Biology; Biotin; Cancer Etiology; Cancer cell line; Cell physiology; Cells; Cellular Stress; Characteristics; Critical Pathways; Development; Differentiation and Growth; Disease; Electrophoretic Mobility Shift Assay; Engineering; Epithelial Cells; Foundations; Gene Activation; Gene Deletion; Gene Duplication; Gene Expression; Gene Expression Regulation; Gene Proteins; Gene Targeting; Genes; Genetic Transcription; Genome; Genotype; HPV-High Risk; Health; High-Risk Cancer; Homeostasis; Human Papilloma Virus Vaccine; Human Papillomavirus; Human papilloma virus infection; Human papillomavirus 16; Immunization; Immunoprecipitation; Incidence; Infection; Intervention; Laboratories; Lead; Link; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Messenger RNA; Methods; Modeling; Morbidity - disease rate; Nucleic Acid Binding; Oncogenic Viruses; Oncoproteins; Pathogenesis; Pathway interactions; Poly(A)-Binding Proteins; Post-Transcriptional Regulation; Preventive vaccine; Process; Proteins; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reagent; Regulation; Ribonucleosides; Risk; Role; TERT gene; Techniques; Technology; Tertiary Protein Structure; Therapeutic; Transcript; Translations; Up-Regulation; Variant; Viral; Viral Oncogene; Virus Diseases; Woman; Work; base; cancer cell; crosslink; duplicate genes; high risk; human model; mRNA Expression; men; mortality; nano-string; new technology; next generation sequencing; novel; overexpression; protein degradation; protein expression; screening; synergism; tool; tumor; uptake

Human papillomavirus (HPV) affects up to 75% of adults and is categorized as high risk (HR) or low risk (LR) based on its association with cancer. Although there are preventive vaccines against specific HPV genotypes, immunizations have had poor uptake and completion. This leaves many women and men at continued risk of HPV infection and HPV-associated cancers, some of which are increasing in incidence. Knowing how HR HPV infections affect their host cells is critical for understanding tumor virus biology and for developing screening and treatments that reduce morbidity and mortality worldwide. Studies in HPV- associated cancers have focused on the malignant potential of HR E6 and E7 viral oncoproteins and their roles in gene activation and protein degradation; however, less well-studied is their role in post-transcriptional cellular gene regulation. We found HR HPV 16E6 (16E6), through its partnership with the endogenous protein NFX1-123, post-transcriptionally upregulated expression of the catalytic subunit of telomerase (hTERT) and a master regulator of growth and differentiation (Notch1). Little is known about the normal function of NFX1-123 in epithelial cells; however, based on the protein domain motifs and partnerships, and cellular localization of NFX1-123, we theorize NFX1-123 participates in RNA binding and regulation in healthy epithelial cells. Indeed, NFX1-123 is needed to increase Notch1 and hTERT mRNA expression by 16E6, and these increases require the NFX1-123 RNA processing protein motifs. This project seeks to identify the global set of mRNAs directly bound by NFX1-123, with and without 16E6, using a novel technique, Photoactivatable-Ribonucleoside- Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), with the depth and breadth of Next Generation Sequencing, and the ease of nanoString Technologies. In Aim 1, we will identify mRNAs directly bound by NFX1-123 in healthy epithelial cells. We hypothesize multiple mRNAs are bound by NFX1-123 as a part of their typical processing and translation, and we have found mRNAs increased with overexpressed NFX1-123 in a large microarray. We will use PAR-CLIP as an unbiased approach to identify novel RNAs bound by NFX1- 123 and to determine whether mRNAs increased in our microarray were due mechanistically to greater amounts of RNA-protein binding by NFX1-123. In Aim 2, we will identify mRNAs bound by NFX1-123 in epithelial cells expressing 16E6, or the full HPV 16 genome, as a model for infected epithelial cells. These two sets of mRNAs bound by NFX1-123 may be mutually exclusive, as HR HPV targets new mRNAs to be bound and upregulated by NFX1-123, or they may overlap in part by HR HPV modulating an mRNA or a pathway that NFX1-123 already typically regulates. The proposed work will use cutting-edge techniques to yield a comprehensive view of post-transcriptional regulation by NFX1-123 and a HR E6 viral oncogene. These studies will lead to new information on mechanisms and pathways critical, and even treatable, in HPV infections and associated cancers.

人乳头瘤病毒（HPV）影响高达75%的成年人，并根据其与癌症的相关性分为高风险（HR）或低风险（LR）。虽然有针对特定HPV基因型的预防性疫苗，但免疫接种的吸收和完成情况很差。这使得许多女性和男性继续面临HPV感染和HPV相关癌症的风险，其中一些癌症的发病率正在增加。了解HR HPV感染如何影响其宿主细胞对于了解肿瘤病毒生物学以及开发降低全球发病率和死亡率的筛查和治疗至关重要。HPV相关癌症的研究集中在HR E6和E7病毒癌蛋白的恶性潜能及其在基因活化和蛋白降解中的作用;然而，对其在转录后细胞基因调控中的作用研究较少。我们发现HR HPV 16 E6（16 E6）通过与内源性蛋白NFX 1 - 123的合作，转录后上调端粒酶催化亚基（hTERT）和生长和分化的主要调节因子（Notch1）的表达。关于NFX 1 - 123在上皮细胞中的正常功能知之甚少;然而，基于NFX 1 - 123的蛋白结构域基序和伙伴关系以及细胞定位，我们推测NFX 1 - 123参与健康上皮细胞中的RNA结合和调节。事实上，需要NFX 1 - 123来增加Notch1和hTERT mRNA表达16E6，并且这些增加需要NFX 1 - 123 RNA加工蛋白基序。该项目旨在确定与NFX 1 - 123直接结合的mRNA的全球集合，有和没有16E6，使用一种新的技术，光活化核糖核苷增强交联和免疫沉淀（PAR-CLIP），具有下一代测序的深度和广度，以及nanoString技术的易用性。在目标1中，我们将鉴定健康上皮细胞中直接与NFX 1 - 123结合的mRNA。我们假设多个mRNA被NFX 1 - 123结合作为其典型加工和翻译的一部分，并且我们在大的微阵列中发现mRNA随着NFX 1 - 123的过表达而增加。我们将使用PAR-CLIP作为一种无偏倚的方法来鉴定NFX 1 - 123结合的新型RNA，并确定我们的微阵列中mRNA的增加是否是由于NFX 1 - 123结合的RNA蛋白量增加所致。在目标2中，我们将鉴定表达16E6或完整HPV 16基因组的上皮细胞中NFX 1 - 123结合的mRNA，作为感染上皮细胞的模型。这两组与NFX 1 - 123结合的mRNA可能是相互排斥的，因为HR HPV靶向与NFX 1 - 123结合并被NFX 1 - 123上调的新mRNA，或者它们可能部分重叠，因为HR HPV调节NFX 1 - 123通常已经调节的mRNA或途径。拟议的工作将使用尖端技术来产生NFX 1 - 123和HR E6病毒癌基因的转录后调控的全面视图。这些研究将为HPV感染和相关癌症的关键机制和途径提供新的信息，甚至是可治疗的。