Best practice standards for circular RNA research

Circular RNA translation: novel protein isoforms and clinical significance.

The design and synthesis of circular RNAs.

Circular RNAs in physiology and non-immunological diseases

Gene expression in eukaryotic cells is a complex process encompassing several layers of regulation at the transcriptional and post-transcriptional levels. At the post-transcriptional level, microRNAs (miRs) are key regulatory molecules that function by binding directly to mRNAs. This generally leads to less efficient translation of the target mRNAs. More recently, an additional layer of gene regulation has been discovered, as other molecules, including circular RNAs (circRNAs), may bind to miRs and thereby function as sponges or decoys resulting in increased expression of the corresponding miR target genes. The circRNAs constitute a large class of mainly non-coding RNAs, which have been extensively studied in recent years, in particular in the cancer research field where many circRNAs have been proposed to function as miR sponges. Here, we briefly describe miR-mediated gene regulation and the extra layer of regulation that is imposed by the circRNAs. We describe techniques and methodologies that are commonly used to investigate potential miR sponging properties of circRNAs and discuss major pitfalls and controversies within this relatively new research field.

Circular RNAs as microRNA sponges: evidence and controversies.

Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.

/pdf/comprehensive-analysis-of-translation-from-overexpressed-4rbve9swva.pdf

Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

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Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.