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Efficient Circularization of Protein-Encoding RNAs via a Novel Cis-Splicing System

ABSTRACT: Circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA, owing to their unique properties and potential therapeutic applications, driving the development of novel approaches for their production. This study introduces a cis-splicing system that efficiently produces circRNAs by incorporating a ribozyme core at one end of the precursor, thereby eliminating the need for additional spacer elements between the ribozyme and the gene of interest (GOI). In this cis-splicing system, sequences resembling homologous arms at both ends of the precursor are crucial for forming the P9.0 duplex, which in turn facilitates effective self-splicing and circularization. We demonstrate that the precise recognition of the second transesterification site depends more on the structural characteristics of P9.0 adjacent to the ωG position than on the nucleotide composition of the P9.0-ωG itself. Further optimization of structural elements, like P10 and P1-ex, significantly improves circularization efficiency. The circRNAs generated through the cissplicing system exhibit prolonged protein expression and minimal activation of the innate immune response. This study provides a comprehensive exploration of circRNA generation via a novel strategy and offers valuable insights into the structural engineering of RNA, paving the way for future advancements in circRNA-based applications.

ORGANISM(S): synthetic construct

PROVIDER: GSE263525 | GEO | 2024/08/21

REPOSITORIES: GEO

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Project description:In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts. A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test. Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.

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Efficient Circularization of Protein-Encoding RNAs via a Novel Cis-Splicing System

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