Noncoding RNAs in gamma-Herpesvirus Biology and AIDS Malignancies

项目概要 卡波西肉瘤相关疱疹病毒 (KSHV) 是一种人类 γ 疱疹病毒，是艾滋病的病原体 KS 和原发性渗出性淋巴瘤 (PEL) 等恶性肿瘤。近年来，人们已经清楚地认识到，致病性 包括 EBV、KSHV 和 MHV68 在内的疱疹病毒表达大量长非编码 RNA (lncRNA) 它们与蛋白质编码转录本呈反义方向。这些RNA的功能和结构是 很大程度上不为人知。此外，这些病毒还表达 microRNA (miRNA)。在表征 KSHV 时 使用改良的杂交交联和测序 (qCLASH) 协议，我们确定了 miRNA 目标组 数百个宿主细胞 lncRNA 作为假定的 miRNA 靶标。这些数据强烈表明 KSHV 编码的蛋白质和 miRNA 会导致宿主 lncRNA 的失调。重要的是，34 个 lncRNA 据报告，KSHV 感染后出现紊乱，包括 MALAT1、HOTTIP、ANRIL、Meg3、UCA1 和 GAS-5 与人类癌症有关。我们还将 mRNA 和 lncRNA 靶点与癌症标志联系起来 表型，如增殖、迁移、血管生成和葡萄糖代谢，并开始识别 作为 KS 模型，人内皮细胞中受 KSHV miRNA 干扰的信号通路。我们也 确定异常剪接是另一种癌症标志表型。在此我们建议延长我们的学习 通过整合来自qCLASH、RNAseq和miRNAseq数据的多组学数据集进行综合分析 KSHV 感染的内皮细胞中 miRNA 调控的基因调控网络。此外，我们建议验证 我们在大量人类肿瘤样本中的发现并通过生成第一个 KS 肿瘤 miRNA qCLASH 的目标组。这些研究将以与项目 2 和 3 进行比较的方式进行 此外，还将得到核心 B、C 和 D 的支持。此外，我们正在功能性地研究 反义 LANA 转录本 (ALT)，我们使用高度测序鉴定了 81 个假定的结合蛋白。 创新的 RNA 下拉测定。此外，我们将研究一类新发现的病毒环状 RNA 的作用 该程序已经发现了这一点。总之，该项目的目标是描绘病毒的作用 艾滋病恶性肿瘤中受病毒感染干扰的 lncRNA 和宿主细胞 lncRNA。

PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is the causative agent of AIDS malignancies like KS and primary effusion lymphomas (PEL). In recent years it became clear that pathogenic herpesviruses including EBV, KSHV, and MHV68 express numerous long non-coding RNAs (lncRNAs) many of which are in antisense orientation to protein coding transcripts. The function and structure of these RNAs is largely unknown. In addition, these viruses express microRNAs (miRNAs). While characterizing the KSHV miRNA targetomes using a modified Crosslinking and Sequencing of Hybrids (qCLASH) protocol, we identified several hundred host cellular lncRNAs as putative miRNA targets. These data strongly suggest that both KSHV encoded proteins and miRNAs contribute to dysregulation of host lncRNAs. Importantly, 34 lncRNAs that are perturbed following KSHV infection, including MALAT1, HOTTIP, ANRIL, Meg3, UCA1 and GAS-5 are reported to be associated with human cancers. We also linked both mRNA and lncRNA targets to cancer hallmark phenotypes such as proliferation, migration, angiogenesis, and glucose metabolism, and started to identify signaling pathways that are perturbed by KSHV miRNAs in human endothelial cells as a model for KS. We also identified aberrant splicing as an additional cancer hallmark phenotype. Here we propose to extend our studies by integrating multi-omics data sets from qCLASH, RNAseq and miRNAseq data to comprehensively analyze miRNA-regulated gene regulatory networks in KSHV infected endothelial cells. Moreover, we propose to validate our findings in a significant number of human tumor samples and by generating the first KS tumor miRNA targetome by qCLASH. These studies will be performed in a comparative fashion with Projects 2 and 3 and furthermore will be supported by Cores B, C, and D. In addition, we are functionally studying the role of the antisense LANA transcript (ALT) for which we have identified 81 putative binding proteins using a highly innovative RNA-pulldown assay. Moreover, we will study the role of a newly identified class of viral circular RNAs that has been discovered by this program. In summary, the goal of this project is to delineate the role of viral lncRNAs and host cellular lncRNAs that are perturbed by viral infection in AIDS malignancies.