The presented work focuses on the design, production, and application of circular RNA (circRNA) for sequestration of RNA-binding protein hnRNP L and modulation of alternative splicing of CD45. For that, we first focused on circRNA expression systems and applied either tRNA-based, Tornado ribozyme-driven or in vitro intron type I-based circRNA expression systems, as well as in vitro-generated circRNAs. In addition, we developed several circRNA expression constructs based on the platelet-specific circRNA Plt-circR4.HnRNP L is a global regulator of alternative splicing, binding preferentially to CA-rich RNA sequences. Therefore, we designed and applied in HeLa cells circRNAs containing CA-rich sequences. Direct in vivo hnRNP L/circRNA interactions were captured by RNA immunoprecipitation (RIP) and alternative splicing of hnRNP L target genes was analyzed by RT-PCR. Interestingly, we observed hnRNP L delocalization from the predominant nuclear localization, which resulted in equal hnRNP L distribution between nucleus and cytoplasm in HeLa cells.As a part of the hnRNP L-regulated alternative splicing network, we targeted the gene coding for CD45, an essential regulator of T- and B-cell antigen receptor signaling. Therefore, we generated in vitro designer antisense circRNAs targeting splice sites, intron, or each of the three variable exons 4, 5, and 6 of the CD45 pre-mRNA as a proof of principle for developing designer antisense circRNAs that function in alternative splicing modulation. Consequently, we co-transfected a CD45 minigene construct together with the antisense circRNAs into HeLa cells and analyzed splicing patterns of CD45 minigene affected by antisense circRNAs. Specific splicing patterns were detected for each antisense circRNA variant applied, and these changes were determined to be at least partially of post-transcriptional nature.Overall, we conclude that designer circRNAs have high potential for modulating activities of RNA-binding proteins or for alterations of particular alternative splicing events. They represent a promising alternative to pharmacological inhibition of proteins and can be applied in a way similar to antisense splice-switching oligonucleotides targeting individual splicing events.
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