RNA circularization strategies in vivo and in vitro

Petkovic, Sonja; Müller, Sabine

doi:10.1093/nar/gkv045

Cited by 263 publications

(225 citation statements)

References 125 publications

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“…During back splicing, the 3 ′ end of a downstream exon is ligated together with the 5 ′ end of an upstream exon of the same molecule (Jeck and Sharpless 2014), and this process is now considered to be an integral, regulated feature of eukaryotic gene expression programs . Despite their ubiquity, the functions of endogenous circRNAs are largely unknown (Hentze and Preiss 2013;Lasda and Parker 2014), and methods for generating and studying them in vivo are few and far between (Ford and Ares 1994;Zhang et al 2014;Petkovic and Müller 2015).…”

Section: Introductionmentioning

confidence: 99%

Metazoan tRNA introns generate stable circular RNAs in vivo

Filonov

Noto

et al. 2015

RNA

165

144

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We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an agedependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science.

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Section: Introductionmentioning

confidence: 99%

Metazoan tRNA introns generate stable circular RNAs in vivo

Filonov

Noto

et al. 2015

RNA

165

144

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“…For example, it has been proposed that many circular RNAs may regulate neuronal functions (Westholm et al 2014;Rybak-Wolf et al 2015;You et al 2015), and artificial circular RNAs containing an IRES (internal ribosome entry site) can be translated (Chen and Sarnow 1995;Wang and Wang 2015). However, the lack of efficient methods for modulating circular RNA levels or ectopically expressing circular RNAs (for review, see Petkovic and Muller 2015) has limited our ability to define functions for these transcripts.…”

mentioning

confidence: 99%

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

et al. 2015

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Thousands of eukaryotic protein-coding genes are noncanonically spliced to produce circular RNAs. Bioinformatics has indicated that long introns generally flank exons that circularize in Drosophila, but the underlying mechanisms by which these circular RNAs are generated are largely unknown. Here, using extensive mutagenesis of expression plasmids and RNAi screening, we reveal that circularization of the Drosophila laccase2 gene is regulated by both intronic repeats and trans-acting splicing factors. Analogous to what has been observed in humans and mice, basepairing between highly complementary transposable elements facilitates backsplicing. Long flanking repeats (∼400 nucleotides [nt]) promote circularization cotranscriptionally, whereas pre-mRNAs containing minimal repeats (<40 nt) generate circular RNAs predominately after 3 ′ end processing. Unlike the previously characterized Muscleblind (Mbl) circular RNA, which requires the Mbl protein for its biogenesis, we found that Laccase2 circular RNA levels are not controlled by Mbl or the Laccase2 gene product but rather by multiple hnRNP (heterogeneous nuclear ribonucleoprotein) and SR (serine-arginine) proteins acting in a combinatorial manner. hnRNP and SR proteins also regulate the expression of other Drosophila circular RNAs, including Plexin A (PlexA), suggesting a common strategy for regulating backsplicing. Furthermore, the laccase2 flanking introns support efficient circularization of diverse exons in Drosophila and human cells, providing a new tool for exploring the functional consequences of circular RNA expression across eukaryotes.

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“…93,94 Methods, such as splint ligation or the use of selfcleaving ribozymes, can also be used to generate circular RNAs in vitro (For a review see ref. 95 ). Beyond allowing ectopic expression of circular RNAs to define their functions, these approaches can be used to design RNA circles that sequester microRNAs or proteins as well as identify novel IRES sequences.…”

Section: Methods For Ectopic Expression Of Circular Rnas In Cellsmentioning

confidence: 99%

Circular RNAs: Unexpected outputs of many protein-coding genes

Wilusz

2016

RNA Biology

111

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Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs, some of which accumulate to higher levels than their associated linear mRNA. Recent work has revealed widespread mechanisms that dictate whether the spliceosome generates a linear or circular RNA. For most genes, circular RNA biogenesis via backsplicing is far less efficient than canonical splicing, but circular RNAs can accumulate due to their long half-lives. Backsplicing is often initiated when complementary sequences from different introns base pair and bring the intervening splice sites close together. This process is further regulated by the combinatorial action of RNA binding proteins, which allow circular RNAs to be expressed in unique patterns. Some genes do not require complementary sequences to generate RNA circles and instead take advantage of exon skipping events. It is still unclear what most mature circular RNAs do, but future investigations into their functions will be facilitated by recently described methods to modulate circular RNA levels.

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RNA circularization strategies in vivo and in vitro

Cited by 263 publications

References 125 publications

Metazoan tRNA introns generate stable circular RNAs in vivo

Metazoan tRNA introns generate stable circular RNAs in vivo

Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins

Circular RNAs: Unexpected outputs of many protein-coding genes

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