Identification and characterization of circular RNAs in differentiation and human disease

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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).