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Inducible Expression of Eukaryotic Circular RNAs from Plasmids
Deirdre C Tatomer1, Dongming Liang1, Jeremy E Wilusz2
1Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, 363 Clinical Research Building, 415 Curie Blvd., Philadelphia, PA, 19104, USA.
Methods in Molecular Biology (Clifton, N.J.)
|August 3, 2017
Summary
Scientists developed a new method to create circular RNAs (circRNAs) using a plasmid. This technique allows for the study and engineering of these stable RNA molecules for various biological applications.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Expression
Background:
- Circular RNAs (circRNAs) are generated from thousands of eukaryotic protein-coding genes through noncanonical splicing.
- circRNAs exhibit enhanced stability against exonucleases compared to linear mRNAs.
- The mechanisms underlying circRNA biogenesis, particularly backsplicing, are increasingly understood.
Purpose of the Study:
- To present a novel plasmid-based method for the ectopic expression of circular RNAs in eukaryotic cells.
- To enable the functional characterization of natural circRNAs through overexpression.
- To facilitate the design and production of artificial circRNAs with specific functionalities.
Main Methods:
- A plasmid system was engineered where the sequence of interest is inserted into an artificial exon flanked by complementary intronic repeats.
- Transfection of the plasmid into eukaryotic cells, followed by induced transcription.
- Cellular splicing machinery catalyzes backsplicing to form circRNAs.
- Northern blot analysis is used to validate the efficiency and specificity of circRNA generation.
Main Results:
- Demonstrated a straightforward method for generating specific circular RNAs in cells.
- Validated the successful production and detection of ectopically expressed circRNAs.
- Showcased the potential for creating designer circRNAs for targeted applications.
Conclusions:
- The developed plasmid-based method provides a powerful tool for studying circular RNA biology.
- This approach allows for the overexpression of natural circRNAs to elucidate their functions.
- Engineered circRNAs can be designed for translation or to interact with specific cellular components like microRNAs and proteins.
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