Production of pure and functional RNA for in vitro reconstitution experiments

Edelmann, F.T.; Niedner, Annika; Niessing, Dierk

doi:10.1016/j.ymeth.2013.08.034

Cited by 36 publications

(26 citation statements)

References 62 publications

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“…For NMR experiments size exclusion chromatography was performed in 50 mM potassium phosphate buffer pH 7.0 and 200 mM NaCl (NMR buffer). Absence of nucleic-acid contamination was confirmed by measuring the ratio of absorption at 260/280 nm ( Edelmann et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Weber

Bao

Hartlmüller

et al. 2016

eLife

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119

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The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.DOI: http://dx.doi.org/10.7554/eLife.11297.001

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Section: Methodsmentioning

confidence: 99%

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Weber

Bao

Hartlmüller

et al. 2016

eLife

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119

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“…RNA was produced by in vitro transcription (E3-118), tRNA scaffold expression, and purification (E3-51 tRNA, E3-118 tRNA, and TAR-57 tRNA) or total chemical synthesis (E3-51; Dharmacon) (38). For in vitro transcription the 118-nt DNA fragment of the ASH1 gene (plasmid pRS405-ASH1 in Table S1) was PCR amplified using primers with the T7 promotor sequence at the 5′ end.…”

Section: Methodsmentioning

confidence: 99%

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast

Niedner

Müller

Moorthy

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs.M essenger RNA localization is a universal feature of eukaryotes (1-3). By complementing transcriptional control (4), it fulfills a variety of functions, including the establishment of cell polarity and specialization of subcellular regions. In recent years, the directional transport of asymmetric synthesis of HO 1 (ASH1) mRNA in budding yeast has emerged as a particularly well-suited model to study mechanistic principles of RNA localization. Here, comparably few proteins participate in the directional transport of ASH1 mRNA and about 30 other transcripts (5, 6).Chromatin-immunoprecipitaton experiments revealed that the dedicated RNA-binding Swi5p-dependent HO expression protein 2 (She2p) binds already cotranscriptionally to nascent ASH1 mRNA (7, 8). Two additional RNA-binding proteins, pumiliohomology domain family protein 6 (Puf6p) and heterogeneous nuclear RNP K-like protein 1 (Khd1p), are also present in the nucleus, bind to ASH1 mRNA, and act in the cytoplasm as translational repressors during ASH1 transport (9-12). A fourth nuclear factor, termed localization of ASH1 mRNA protein 1 (Loc1p), has been implicated in the assembly of nuclear premessenger ribonucleoprotein particles (mRNPs). Like Puf6p, Loc1p is a nuclear protei...

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“…In vitro transcription is often associated with terminal heterogeneity, dramatically decreasing the yield of ligation for circularization. This can be overcome by the use of special protocols ( 60 ). However, circularization of larger RNAs remains challenging.…”

Section: Circularization In Vitromentioning

confidence: 99%

RNA circularization strategies in vivo and in vitro

Petkovic

Müller

2015

Nucleic Acids Research

256

218

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In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.

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Production of pure and functional RNA for in vitro reconstitution experiments

Cited by 36 publications

References 62 publications

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast

RNA circularization strategies in vivo and in vitro

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