Functions of circular RNAs generated from backsplicing of the HIV-1 primary transcript

项目总结

PROJECT SUMMARY Circular RNAs (circRNAs) are a family of non-coding RNAs that originate from a non-canonical splicing event, named backsplicing, in which the upstream and downstream splicing sites of an RNA transcript become covalently linked to form a closed loop of RNA as opposed to the typical linear structure of mRNA transcripts. Cellular circRNAs play a role in development, cell differentiation, proliferation and are associated with cancer, neurological disorders, autoimmune and cardiovascular disease. circRNAs act as miRNAs sponges, thus inhibiting their function, and by interacting with RNA binding proteins (RBPs), modulating their availability, localization and activity. A few circRNAs have been also shown to modulate transcription and can be translated into short proteins. CircRNAs of viral origin have been identified in DNA viruses from the Herpesviridae and Papillomaviridae families. These viral circRNAs have been shown to promote cell proliferation and metastasis by acting as sponges for miRNAs with functions in tumor suppression. To date, no circRNA encoded by an RNA virus has been characterized. Analysis of the complex HIV-1 splicing pattern revealed that this virus has the potential to generate at least 15 distinct circRNAs. We designed several sets of divergent PCR primers to specifically amplify the predicted HIV circRNA isoforms. Divergent primers were designed to extend in opposite directions on templates from linear transcripts but extend towards each and amplify circularized transcripts. We were able to amplify and confirm by sequencing 15 distinct circRNA isoforms generated from backsplicing of the viral pre-mRNA. Given the long half-life of circRNAs (4-5 times the one of linear RNAs) it is plausible that HIV derived circRNAs modulate viral replication and infection by conditioning the infected cells. In this study we propose to define the precise amount and composition of the circular RNA molecules produced by the virus in primary T cells and a HIV latency model utilizing a combination of circRNA isoform specific qPCR and RNA Sequencing assays. We will also determine the role of the HIV-1 circRNAs in viral replication utilizing both, gain and loss of functions assays.