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）影响多达75%的成年人，根据其与癌症的关系分为高风险（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度都很低。这使得许多女性和男性持续面临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。了解人乳头瘤病毒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筛查和治疗方法以降低全球发病率和死亡率至关重要。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 HPV 16E6 （16E6）通过其与内源性蛋白NFX1-123的伙伴关系，转录后上调端粒酶催化亚基（hTERT）和生长和分化的主要调节剂（Notch1）的表达。关于NFX1-123在上皮细胞中的正常功能知之甚少；然而，基于NFX1-123的蛋白结构域基序和伙伴关系，以及NFX1-123的细胞定位，我们推测NFX1-123在健康上皮细胞中参与RNA结合和调控。事实上，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加工蛋白基序。该项目旨在利用下一代测序的深度和广度，以及纳米链技术的易用性，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），识别直接与NFX1-123结合的mrna的全球集合，无论是否含有16E6。在Aim 1中，我们将在健康上皮细胞中鉴定由NFX1-123直接结合的mrna。我们假设多个mrna被NFX1-123结合，作为其典型加工和翻译的一部分，并且我们在大型微阵列中发现mrna随着NFX1-123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NFX1-123结合的新型rna，并确定我们的微阵列中mrna的增加是否由于NFX1-123结合的rna -蛋白的数量增加。在Aim 2中，我们将在表达16E6或完整HPV 16基因组的上皮细胞中鉴定由NFX1-123结合的mrna，作为感染上皮细胞的模型。这两组由NFX1-123结合的mRNA可能是相互排斥的，因为HR HPV靶向新的mRNA被NFX1-123结合并上调，或者它们可能部分重叠，因为HR HPV调节了NFX1-123通常调节的mRNA或途径。这项工作将使用尖端技术来全面了解NFX1-123和一个hre6病毒致癌基因的转录后调控。这些研究将带来关于HPV感染和相关癌症至关重要甚至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.