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Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA

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Circular RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1724))

Abstract

Human transcriptome contains a large number of circular RNAs (circRNAs) that are mainly produced by back splicing of pre-mRNA. Here we describe a minigene reporter system containing a single exon encoding split GFP in reverse order, which can be efficiently back spliced to produce a circRNA encoding intact GFP gene. This simple reporter system can be adopted to study how different cis-elements and trans-factors affect circRNA production, and also can serve as a reliable system to measure the activity of IRES-mediated translation. Therefore this system can serve as a platform for mechanistic studies on the circRNA biogenesis and its function.

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References

  1. Wang Z, Burge CB (2008) Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA 14:802–813. https://doi.org/10.1261/rna.876308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Chen LL (2016) The biogenesis and emerging roles of circular RNAs. Nat Rev Mol Cell Biol 17:205–211. https://doi.org/10.1038/nrm.2015.32

    Article  CAS  PubMed  Google Scholar 

  3. Chen CY, Sarnow P (1995) Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268:415–417

    Article  CAS  PubMed  Google Scholar 

  4. Wang Y, Wang Z (2015) Efficient backsplicing produces translatable circular mRNAs. RNA 21:172–179. https://doi.org/10.1261/rna.048272.114

    Article  PubMed  PubMed Central  Google Scholar 

  5. Yang Y, Fan X, Mao M, Song X, Wu P, Jin Y, Yang Y, Wong C, Xiao X, Wang Z (2017) Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res 27:626–641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wang Y, Ma M, Xiao X, Wang Z (2012) Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules. Nat Struct Mol Biol 19:1044–1052. https://doi.org/10.1038/nsmb.2377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wang Y, Wang Z (2014) Systematical identification of splicing regulatory cis-elements and cognate trans-factors. Methods 65:350–358. https://doi.org/10.1016/j.ymeth.2013.08.019

    Article  CAS  PubMed  Google Scholar 

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Correspondence to Zefeng Wang .

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Yang, Y., Wang, Z. (2018). Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA. In: Dieterich, C., Papantonis, A. (eds) Circular RNAs. Methods in Molecular Biology, vol 1724. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7562-4_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7562-4_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7561-7

  • Online ISBN: 978-1-4939-7562-4

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