Identification and characterization of circular RNAs in differentiation and human disease

RNA生物学的最新进展引起了人们对一类未被探索的非编码RNA的关注，这些非编码RNA对应于剪接转录的环状异构体。动物细胞表达数以千计的CircRNA，其表达水平与mRNAs相当，通常是以组织或发育阶段特有的方式。CircRNA对核酸外切酶具有天然的抵抗力，因此在细胞内外高度稳定。新出现的证据表明，CircRNA在转录后调控中发挥作用；然而，它们与人类发育和疾病的相关性尚不清楚。我们将检验CircRNAs可能是分化或疾病的合适生物标记物的假设。我们将从健康供者和患者的血液、唾液、尿液和间充质干细胞(MSC)中鉴定CircRNA。我们将研究MSC分化为脂肪(代谢综合征)、血管平滑肌(高血压)、骨或软骨(骨骼表型)前后的情况。我们还将研究感染性疾病和癌症中的CircRNA。具体地说，我们已经建立了合作关系，以研究结肠癌和胰腺癌(柏林Charité综合癌症中心)、Burkitt淋巴瘤和感染EBV的免疫细胞、卡波西肉瘤相关HHV-8和HHV-7(Pfeffer实验室，美国斯特拉斯堡)或艾滋病毒和结核分枝杆菌(Goldfeld实验室，哈佛医学院)。这份由拉杰斯基实验室(MDC柏林-布赫)和洛夫特实验室(夏里特-柏林医科大学，洪堡大学)联合提出的建议。有五个目标。目的1：建立一种从不同组织和样本中分离纯化核糖体RNA缺失转录本和CircRNA并进行深度测序的标准化方法。目的2：开发一种标准化的计算流水线，能够以高通量的方式映射、分析和注释CircRNA序列数据。目的3：鉴定和鉴定原代人骨髓间充质干细胞和骨髓间充质干细胞分化的细胞类型的CircRNA，以深入了解CircRNA在组织发育和永生化中的作用。目的：从临床相关的人体标本(包括病毒感染组织和肿瘤)中鉴定和研究CircRNA，以阐明CircRNA作为诊断和治疗干预的生物标志物的潜力。目标5：通过Dorina公开所有协议、计算代码和测序数据，Dorina是一个可在dorina.mdc-berlin.de上免费访问的经过管理的数据库。我们预计，作为社区资源，该项目将1)提供协议、计算代码以及来自不同组织、病毒感染细胞和临床样本的全面的核糖体RNA耗尽转录组数据；2)识别和注释相应的CircRNA数据集；3)具体识别与分化和疾病状态相关的人类或病毒衍生的CircRNA；4)优先考虑CircRNA用于未来的功能或翻译后续研究(其本身不在本提案的范围内)。

Recent advances in RNA biology have drawn attention to an unexplored class of non-coding RNAs that correspond to circular isoforms of spliced transcripts. Animal cells express thousands of circRNAs at levels comparable to mRNAs, often in a tissue- or developmental stage-specific way. CircRNAs are naturally exonuclease-resistant, and thus highly stable - inside and outside of cells. Emerging evidence suggests a role for circRNAs in post-transcriptional regulation; however, their relevance to human development and disease is unknown. We will test the hypothesis that circRNAs may be suitable biomarkers for differentiation or disease. We will identify circRNAs in human blood, saliva, urine, and mesenchymal stem cells (MSC) from healthy donors and patients. We will investigate MSC before and after differentiation into fat (metabolic syndrome), vascular smooth muscle (hypertension), bone or cartilage (skeletal phenotypes). We will also investigate circRNAs in infectious disease and cancer.Specifically, we have established collaborations to investigate colon and pancreatic cancers (Charité Comprehensive Cancer Center, Berlin), Burkitt lymphoma and immune cells infected with EBV, Kaposi sarcoma-associated HHV-8, and HHV-7 (Pfeffer laboratory, U. Strasbourg) or HIV and M. tuberculosis (Goldfeld laboratory, Harvard Medical School). This joint proposal from the Rajewsky laboratory (MDC Berlin-Buch) and the Luft laboratory (Charité - Medical University Berlin, Humboldt U.) has five aims. Aim 1: To establish a standardized protocol for purification and deep sequencing of ribosomal RNA-depleted transcriptomes and circRNAs from different tissue and sample types. Aim 2: To develop a standardized computational pipeline that can map, analyze and annotate circRNA sequence data in a high-throughput manner. Aim 3: To identify and characterize circRNAs from primary human MSC and MSC-derived, differentiated cell types in order to gain insight into the role of circRNAs during tissue development and perpetuation. Aim 4: To identify and investigate circRNAs from clinically relevant human samples, including virally infected tissues and tumors, in order to elucidate the potential of circRNAs as biomarkers for diagnosis and therapeutical interventions. Aim 5: To make all protocols, computational codes and sequencing data publicly available via doRiNA, a curated database freely accessible at dorina.mdc-berlin.de. We expect that this project will 1) provide - as a community resource - protocols, computational code, as well as comprehensive ribosomal RNA-depleted transcriptome data from different tissues, virally infected cells and clinical samples; 2) identify and annotate corresponding circRNA data sets; 3) specifically identify human or virus-derived circRNAs associated with differentiation and disease states; 4) prioritize circRNAs for future functional or translational follow-up studies (which are themselves beyond the scope of this proposal).