A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing

Schmidt, Casey A.; Noto, John J.; Filonov, Grigory S.; Matera, A. Gregory

doi:10.1016/bs.mie.2016.02.018

Cited by 28 publications

(42 citation statements)

References 22 publications

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“…Primers used to make PCR products for in vitro transcription (to make dsRNA) can be found in Table S4. RNA was isolated using TRIzol Reagent (Invitrogen), with a second chloroform extraction and ethanol rather than isopropanol precipitation (48). To test knockdown efficiency, total RNA was treated with TURBO DNase (Invitrogen) and then converted to cDNA using the SuperScript III kit (Invitrogen) with random hexamer priming.…”

Section: Drosophila Cell Culture Rnai and In-gel Stainingmentioning

confidence: 99%

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Hayne

Schmidt²,

Matera³

et al. 2019

Preprint

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The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.We thank Drs. Andrew Sikkema and Marcos Morgan as well as members of the Stanley Lab for their critical reading of this manuscript. We would like to acknowledge Robert Petrovich and the NIEHS Protein Expression Facility for assistance with protein expression in HEK293 suspension cultures and for supplying the anti-GFP resin. We also thank Robert Dutcher for his assistance with running SEC-MALS.

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Section: Drosophila Cell Culture Rnai and In-gel Stainingmentioning

confidence: 99%

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Hayne

Schmidt²,

Matera³

et al. 2019

Preprint

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“…The human and Drosophila tricRNA reporters used in this study were made previously (9). To generate the dual reporter, four point mutations were introduced in the 3' exon of the Drosophila tricRNA reporter using Q5 Site-Directed Mutagenesis (NEB).…”

Section: Generation Of Reporter and Mutant Constructsmentioning

confidence: 99%

Molecular determinants of metazoan tricRNA biogenesis

Schmidt

Giusto

Matera

2018

Preprint

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Mature tRNAs are generated by multiple post-transcriptional processing steps, which can include intron removal. Recently, our laboratory discovered a new class of metazoan circular RNAs formed by ligation of excised tRNA introns; we termed these molecules tRNA intronic circular (tric)RNAs. To investigate the mechanism of tricRNA biogenesis, we generated constructs that replace the native introns of two Drosophila tRNA genes with the Broccoli fluorescent RNA aptamer. Using these reporters, we identified cis-acting elements required for tricRNA formation in both human and fly cells.We observed that disrupting the conserved anticodon-intron base pair dramatically reduces tricRNA levels. Although the integrity of this base pair is necessary for proper splicing, it is not sufficient.Furthermore, we found that strengthening weak base pairs in the pre-tRNA also impairs tricRNA production. We also used the reporters to identify trans-acting tricRNA processing factors. We found that several known tRNA processing factors, such as RtcB ligase and components of the TSEN endonuclease complex, are involved in tricRNA biogenesis. Depletion of these factors inhibits tRNA intron circularization. Furthermore, we observed that depletion of Clipper endonuclease results in increased tricRNA levels. In summary, our work characterizes the major players in Drosophila tricRNA biogenesis.

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“…Robust expression can be achieved with an external pol III promoter, such as the one from the U6 snRNA gene. 10,54 Pol III transcription can exceed that of pol II, and produces more consistent levels of expression across cell types as compared with the CMV promoter. 55,56 Accurate characterization of circRNA expression can be difficult, however, due to a wide variety of circRNA sizes.…”

Section: Methods Of Ectopic Circrna Expression and Detectionmentioning

confidence: 99%

“…However, RT-PCR of a short circRNA can produce concatemers that are many times longer than the original transcript. 54 Thus, circRNA length must be carefully considered when using RT-based methods to quantify transcript abundance, and direct measurement of circRNA abundance by fluorescence or northern blotting may be preferred. We have successfully expressed 'designer 0 tricRNAs greater than 800 nt in length (Fig.…”

Section: Methods Of Ectopic Circrna Expression and Detectionmentioning

confidence: 99%

Engineering and expressing circular RNAs via tRNA splicing

2017

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Circular (circ)RNAs have recently become a subject of great biologic interest. It is now clear that they represent a diverse and abundant class of RNAs with regulated expression and evolutionarily conserved functions. There are several mechanisms by which RNA circularization can occur in vivo. Here, we focus on the biogenesis of tRNA intronic circular RNAs (tricRNAs) in archaea and animals, and we detail their use as research tools for orthogonal, directed circRNA expression in vivo.

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A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing

Cited by 28 publications

References 22 publications

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

Molecular determinants of metazoan tricRNA biogenesis

Engineering and expressing circular RNAs via tRNA splicing

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