随着高通量测序技术的不断完善，越来越多的非编码RNA被鉴定出来，其中环状RNA也成为一个研究重点，但有关环状RNA的调控机制尚不清楚。我们前期研究利用报告基因系统证实了环状RNA可以通过反向剪接产生，并能够翻译成功能性蛋白（RNA，2015）。本研究将在前期工作基础上，首先采用本实验室经典的GFP报告基因系统与高通量测序技术相结合的方法，系统性研究调控环状RNA生成的顺式作用元件并验证其功能。其次，利用亲和层析与蛋白质谱技术相结合的方法，鉴定与环状RNA顺式作用元件相结合的蛋白剪接因子，并验证其功能及功能结构域。在此基础上，我们将利用环状RNA双色报告基因系统及高内涵细胞分析仪，鉴定调控环状RNA生成的小分子化合物，并对其功能和调控机制进行研究。本项目的顺利实施将进一步完善调控环状RNA生成的分子机制，并为今后利用小分子化合物调控内源性环状RNA的生成及功能提供可能性和更完善的实验依据。

With the application of the next generation sequencing, a number of non-coding RNAs have been identified, including circular RNAs. However, the mechanisms of how circular RNAs are generated and their regulatory rules still remain largely unknown. Previously we constructed a single exon mini-gene reporter containing split GFP, and found that the pre-mRNA indeed produces circular RNAs through efficient back-splicing in human and Drosophila. Importantly, the resulting circular RNAs can be translated to generate functional proteins. Here we propose to systematically identify the cis-elements that regulate the biogenesis of circular RNA using GFP mini-gene reporter and next generation sequencing approaches. Subsequently, we will identify protein factors that bind to these cis-elements with affinity purification and mass spectrometry approaches, and further validate the functions of the identified splicing factors. Moreover, we will combine dual-color circular RNA reporter with BD pathway system to identify small chemicals that can regulate circular RNA biogenesis and functions. This project will partially uncover the regulatory mechanisms of circular RNA biogenesis, and provide experimental evidence to study the regulatory roles of small chemicals on circular RNA biogenesis. More importantly, this study provides great therapeutic potentials for targeting circular RNA related diseases in the future.