In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.
|Journal||Journal of Visualized Experiments|
|State||Published - Mar 2020|
Bibliographical noteFunding Information:
This work was supported by the Department of Defense DoD grant AZ180075. Stefan Stamm thanks Jacqueline Noonan Endowment. Anna Pawluchin was supported by the DAAD, German academic exchange program, Justin R. Welden was a recipient of the University of Kentucky Max Steckler Award.
© 2020 Journal of Visualized Experiments.
- Alternative splicing
- Circular RNAs
- Issue 157
- Reporter gene
ASJC Scopus subject areas
- Neuroscience (all)
- Chemical Engineering (all)
- Biochemistry, Genetics and Molecular Biology (all)
- Immunology and Microbiology (all)