Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

Jiang, Yanyi; Chen, Xiaofan; Zhang, Wei

doi:10.1016/j.bbrc.2021.04.044

Cited by 9 publications

(6 citation statements)

References 40 publications

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“…Notably, two studies recently reported that the backsplicing system expresses several linear transcripts (Ho-Xuan et al, 2020;Jiang et al, 2021). Thus, the backsplicing system produces most of its RNA in a linear form while the Tornado system produces predominantly circular mRNA.…”

Section: Resultsmentioning

confidence: 99%

“…However, our experiments showed that the predominant product of this reaction was a linear product, likely reflecting the forward splicing reaction. Other groups similarly found that the backsplicing system generated linear forward splicing products (Ho-Xuan et al, 2020;Jiang et al, 2021). In contrast, the Tornado system readily generated large circular mRNAs (up to 4719 bp), with minimal detectable linear precursors.…”

Section: Discussionmentioning

confidence: 97%

“…In order to achieve this, circular mRNAs need to be generated in cells and then packaged into VLPs. Previous work has found that the standard method for creating large circular RNAs, termed the backsplicing system (Liang and Wilusz, 2014), generates linear RNA species and would therefore be an inefficient method for producing VLPs that contain a circular mRNA (Ho-Xuan et al, 2020;Jiang et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

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Highly efficient cellular expression of circular mRNA enables prolonged protein expression

Unti

Jaffrey

2023

Preprint

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A major problem with mRNA therapeutics is that the mRNA is usually degraded within a few hours after entering the cytosol. This limits the duration and total amount of protein expression. New approaches for in vitro synthesis of circular mRNA have allowed increased levels and duration of protein synthesis from mRNA therapeutics due to the long half-life of circular mRNA. However, it remains difficult to genetically encode circular mRNAs in mammalian cells, which makes it difficult to use circular mRNA in cell-derived therapeutics. Here we describe the adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system to achieve in-cell synthesis of circular mRNAs. We screen different promoters and internal ribosomal entry sites (IRESs), and identify combinations that result in high levels of circular mRNA and protein expression in cells. We show that these circular mRNAs can be packaged into virus-like particles (VLPs) thus enabling prolonged protein expression. Overall, these data describe a platform for synthesis of circular mRNAs and how these circular mRNAs can markedly enhance the ability of VLPs to function as a mRNA delivery tool.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Highly efficient cellular expression of circular mRNA enables prolonged protein expression

Unti

Jaffrey

2023

Preprint

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“…For instance, CircE7, which was identified in papillomaviruses, can be translated despite the lack of an ATG start codon 104 . Circ-PAPOLA, lacking an annotated AUG start codon, is also translated 105 . Considering the non-AUG translation initiation observed for circRNAs, it is plausible that other phenomena that occur during the translation of linear mRNAs, such as ribosome read-through and reinitiation, could also occur during circRNA translation.…”

Section: Translation Start and Stop On Circrnasmentioning

confidence: 99%

Molecular mechanisms of circular RNA translation

Hwang,

Kim

2024

Exp Mol Med

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Circular RNAs (circRNAs) are covalently closed single-stranded RNAs without a 5′ cap structure and a 3′ poly(A) tail typically present in linear mRNAs of eukaryotic cells. CircRNAs are predominantly generated through a back-splicing process within the nucleus. CircRNAs have long been considered non-coding RNAs seemingly devoid of protein-coding potential. However, many recent studies have challenged this idea and have provided substantial evidence that a subset of circRNAs can associate with polysomes and indeed be translated. Therefore, in this review, we primarily highlight the 5’ cap-independent internal initiation of translation that occurs on circular RNAs. Several molecular features of circRNAs, including the internal ribosome entry site, N6-methyladenosine modification, and the exon junction complex deposited around the back-splicing junction after back-splicing event, play pivotal roles in their efficient internal translation. We also propose a possible relationship between the translatability of circRNAs and their stability, with a focus on nonsense-mediated mRNA decay and nonstop decay, both of which are well-characterized mRNA surveillance mechanisms. An in-depth understanding of circRNA translation will reshape and expand our current knowledge of proteomics.

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“…Mechanistically, circRNAs often function as miRNA sponges with gene-regulatory potential ( Huang et al, 2020 ). CircRNAs can also serve as scaffolds to enhance the interaction between target proteins ( Wang et al, 2019 ; Li et al, 2021 ) or translatable transcripts that can produce protein isoforms ( Liang et al, 2019 ; Jiang et al, 2021 ). MiRNA sponges are the most prevailing mechanism for the functions of circRNAs.…”

Section: Introductionmentioning

confidence: 99%

CircFGGY Inhibits Cell Growth, Invasion and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma via Regulating the miR-545-3p/Smad7 Axis

Feng

Diao

Zhou

et al. 2022

Front. Cell Dev. Biol.

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Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Circular RNAs (circRNAs) play critical roles in the progression of HCC. However, the role of the newly identified circFGGY (hsa_circ_0006633) in the development and progression of HCC has not been explored. In this study, we found that circFGGY was significantly downregulated in tumor compared with that in adjacent normal liver tissues of patients with HCC. HCC patients with low circFGGY expression had poor overall survival after hepatectomy. Moreover, it was found that circFGGY could inhibit the proliferation, invasion and epithelial-mesenchymal transition of HCC both in vivo and in vitro. Mechanistically, circFGGY promoted the expression of Smad7, a well-known suppressor of the transforming growth factor-β signaling pathway. In addition, miR-545-3p, a tumor promoter targeting both circFGGY and Smad7, suppressed the upregulation of Smad7 caused by circFGGY overexpression. Collectively, our data revealed that circFGGY inhibits the proliferation and invasion of HCC cells by sponging miR-545-3p and promote the expression of Smad7, indicating that circFGGY functions as a tumor suppressor and could be a prognostic biomarker for HCC.

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Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

Cited by 9 publications

References 40 publications

Highly efficient cellular expression of circular mRNA enables prolonged protein expression

Highly efficient cellular expression of circular mRNA enables prolonged protein expression

Molecular mechanisms of circular RNA translation

CircFGGY Inhibits Cell Growth, Invasion and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma via Regulating the miR-545-3p/Smad7 Axis

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