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

人类乳头瘤病毒(HPV)影响高达75%的成年人，根据其与癌症的关系被归类为高风险(HR)或低风险(LR)。尽管有针对特定HPV基因类型的预防性疫苗，但免疫接种的接受率和完成率很低。这使许多女性和男性继续面临感染HPV和HPV相关癌症的风险，其中一些癌症的发病率正在上升。了解HR HPV感染如何影响宿主细胞，对于了解肿瘤病毒生物学以及开发可在全球范围内降低发病率和死亡率的筛查和治疗方法至关重要。HPV相关癌症的研究主要集中在HR E6和E7病毒癌蛋白的恶性潜能及其在基因激活和蛋白质降解中的作用；然而，对它们在转录后细胞基因调控中的作用研究较少。我们发现HR HPV16E6(16E6)通过与内源性蛋白NFX1-123配对，转录后上调端粒酶催化亚单位(HTERT)的表达，并作为生长和分化的主要调节因子(Notch1)。我们对NFX1-123在上皮细胞中的正常功能知之甚少；然而，根据NFX1-123的蛋白质结构域和伙伴关系以及细胞定位，我们推测NFX1-123参与了健康上皮细胞的RNA结合和调节。的确，NFX1-123需要将Notch1和hTERT mRNA的表达增加16E6，而这些增加需要NFX1-123 RNA处理蛋白基序。该项目试图利用一种新的技术--光激活-核糖核苷增强的交联和免疫沉淀(PAR-CLIP)，结合下一代测序的深度和广度，以及纳米串技术的简便性，识别直接与NFX1-123结合的全球mRNAs集，包括16E6和不包括16E6的mRNA。在目标1中，我们将在健康的上皮细胞中鉴定与NFX1-123直接结合的mRNAs。我们假设多个mRNAs被NFX1-123结合作为其典型处理和翻译的一部分，我们发现在大型微阵列中，mRNAs随着NFX1-123的过度表达而增加。我们将使用PAR-CLIP作为一种无偏倚的方法来识别与NFX1-123结合的新RNA，并确定我们的微阵列中增加的mRNAs是否机械地归因于NFX1-123与更多的RNA-蛋白质结合。在目标2中，我们将在表达16E6或HPV16全基因组的上皮细胞中鉴定与NFX1-123结合的mRNAs，作为感染上皮细胞的模型。这两组由NFX1-123结合的mRNAs可能是相互排斥的，因为HR HPV靶向NFX1-123结合和上调的新mRNAs，或者它们可能部分重叠，通过HR HPV调节NFX1-123已经典型地调节的mRNA或途径。这项拟议的工作将使用尖端技术来全面了解NFX1-123和HR E6病毒癌基因的转录后调控。这些研究将带来关于HPV感染和相关癌症中关键的、甚至是可治疗的机制和途径的新信息。

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.