In vitro Generation of a Circular Exon from a Linear Pre-mRNA Transcript

Schindewolf, Catherine; Braun, Susanne; Domdey, Horst

doi:10.1093/nar/24.7.1260

Cited by 47 publications

(36 citation statements)

References 39 publications

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“…CircRNAs are generally formed by alternative splicing of pre-mRNA ( Fig. 1), in which an upstream splice acceptor is joined to a downstream splice donor in a process known as 'backsplicing' (Barrett et al, 2015;Schindewolf et al, 1996;Starke et al, 2015). Although there is still no consensus as to the function of circRNAs, a number of studies have revealed that circRNAs are expressed in a variety of eukaryotic organisms, demonstrate conservation across mammals, and are expressed in a regulated manner independent of their cognate linear isoforms.…”

Section: Introductionmentioning

confidence: 99%

Circular RNAs: analysis, expression and potential functions

2016

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Just a few years ago, it had been assumed that the dominant RNA isoforms produced from eukaryotic genes were variants of messenger RNA, functioning as intermediates in gene expression. In early 2012, however, a surprising discovery was made: circular RNA (circRNA) was shown to be a transcriptional product in thousands of human and mouse genes and in hundreds of cases constituted the dominant RNA isoform. Subsequent studies revealed that the expression of circRNAs is developmentally regulated, tissue and cell-type specific, and shared across the eukaryotic tree of life. These features suggest important functions for these molecules. Here, we describe major advances in the field of circRNA biology, focusing on the regulation of and functional roles played by these molecules.

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Section: Introductionmentioning

confidence: 99%

Circular RNAs: analysis, expression and potential functions

2016

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“…Alternatively, the splicing machinery can ''backsplice'' and join a splice donor to an upstream splice acceptor, thereby yielding a circular RNA that has covalently linked ends. Supporting this biogenesis model, canonical splicing signals generally immediately flank regions that circularize, and exon circularization can be observed when splicing substrates are incubated in mammalian nuclear extracts Pasman et al 1996;Schindewolf et al 1996). Once generated, circular RNAs appear to almost always be noncoding, as the genic start and/or stop codons have been removed.…”

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confidence: 97%

Short intronic repeat sequences facilitate circular RNA production

2014

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Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from proteincoding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery ''backsplices'' and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (~30-to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 39 end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA.

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“…However, circular, single-exon-containing RNA molecules have also been observed during the expression of the ETS-1 gene (1). In addition, it has been shown that circular RNA molecules can be readily generated in vitro from both linear or circular yeast pre-mRNAs (18,19).…”

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confidence: 99%

Exon Skipping and Circular RNA Formation in Transcripts of the Human Cytochrome P-450 2C18 Gene in Epidermis and of the Rat Androgen Binding Protein Gene in Testis

Zaphiropoulos

1997

Molecular and Cellular Biology

159

139

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The cytochrome P-450 2C18 gene was found by reverse transcription-PCR to represent the most abundantly expressed gene of the P-450 2C subfamily in human epidermis. However, in addition to the canonical mRNA of nine exons, transcripts that have skipped exon 4 or 5, exons 4, 5, and 6, or exons 4, 5, 6, and 7 were also identified in this tissue. Remarkably, circular RNA transcripts synthesized by the joining of the donor and acceptor splice sites of the same exon were detected in human epidermis for exons 4 and 5. Moreover, molecules composed of exons 4, 5, and 6 with the donor splice site of exon 6 joined to the acceptor splice site of exon 4 or composed of exons 4, 5, 6, and 7 with the donor splice site of exon 7 joined to the acceptor splice site of exon 4 were also found to be present in this tissue. In rat testis, a similar analysis allowed the detection of a circular RNA molecule composed of exons 6 and 7 of the androgen binding protein (ABP) gene, with the donor splice site of exon 7 joined to the acceptor splice site of exon 6, and of an ABP mRNA which had skipped exons 6 and 7. These results apparently substantiate the hypothesis that alternative pre-mRNA splicing has the potential to generate not only mRNAs that lack one or more exons but also circular RNA molecules that are composed of the exons that are skipped. However, additional 2C18 circular species containing various combinations of exons were also detected in human epidermis, and an exon 6-skipped ABP mRNA molecule was identified in rat testis. This observation is interpreted as indicative that at low frequency, numerous circular RNA formation and exon skipping events may occur, allowing the joining of a variety of different combinations of exons. Moreover, the relative stability of these molecules is apparently the key factor that determines the relative ease of their detection.

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In vitro Generation of a Circular Exon from a Linear Pre-mRNA Transcript

Cited by 47 publications

References 39 publications

Circular RNAs: analysis, expression and potential functions

Circular RNAs: analysis, expression and potential functions

Short intronic repeat sequences facilitate circular RNA production

Exon Skipping and Circular RNA Formation in Transcripts of the Human Cytochrome P-450 2C18 Gene in Epidermis and of the Rat Androgen Binding Protein Gene in Testis

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