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Pathways for macrophage uptake of cell-free circular RNAs

ABSTRACT: Pathways for macrophage uptake of cell-free circular RNAs

PROVIDER: PRJNA1101075 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28709800_1.fastq.gz	Fastqsanger.gz
	SRR28709800_2.fastq.gz	Fastqsanger.gz
	SRR28709801_1.fastq.gz	Fastqsanger.gz
	SRR28709801_2.fastq.gz	Fastqsanger.gz
	SRR28709802_1.fastq.gz	Fastqsanger.gz

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Pathways for macrophage uptake of cell-free circular RNAs

Project description:Circular RNAs (CircRNAs) are stable RNAs present in cell-free RNA, comprising cellular debris and pathogen genomes. As a prerequisite for the development of efficient circRNA-based therapies, we investigated the phenomenon and mechanism of cellular uptake and intracellular fate of circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid, energy-dependent, and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake, with distinct gene expression programs depending on the mechanism involved. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1, toll-like receptors, and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs, a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an "eat me" signal and a danger-associated molecular pattern, indicating orderly pathways of recognition and disposal.

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Project description:Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq

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Project description:Natural circular RNAs have been found to sequester microRNAs and suppress their function. We have used this principle as a molecular tool, and produced artificial circular RNA sponges in a cell-free system by in vitro transcription and ligation. Formerly, we were able to inhibit hepatitis C virus propagation by applying a circular RNA decoy strategy against microRNA-122, which is essential for the viral life cycle. In another proof-of-principle study, we used circular RNAs to sequester microRNA-21, an oncogenic and pro-proliferative microRNA. This strategy slowed tumor growth in a 3D cell culture model system, as well as in xenograft mice upon systemic delivery. In the wake of the global use of an in vitro transcribed RNA in Covid-19 vaccines, the question arose if therapeutic circular RNAs trigger cellular antiviral defense mechanisms when delivered systemically. In this study, we present data on the cellular innate immune response as a consequence of liposome-based transfection of the circular RNA sponges we previously used to inhibit microRNA function. We find that circular RNAs produced by the presented methodology do not trigger the antiviral response and do not activate innate immune signaling pathways.

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