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RNA biology in a test tube—an overview of in vitro systems/assays

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Abstract In vitro systems have provided a wealth of information in the field of RNA biology, as they constitute a superior and sometimes the unique approach to address many important questions. Such cell‐free methods can be sorted by the degree of complexity of the preparation of enzymatic and/or regulatory activity. Progress in the study of pre‐mRNA processing has largely relied on traditional in vitro methods, as these reactions have been recapitulated in cell‐free systems. The pre‐mRNA capping, editing, and cleavage/polyadenylation reactions have even been reconstituted using purified components, and the enzymes responsible for catalysis have been characterized by such techniques. In vitro splicing using nuclear or cytoplasmic extracts has yielded clues on spliceosome assembly, kinetics, and mechanisms of splicing and has been essential to elucidate the function of splicing factors. Coupled systems have been important to functionally connect distinct processes, like transcription and splicing. Extract preparation has also been adapted to cells from a variety of tissues and species, revealing general versus species‐specific mechanisms. Cell‐free assays have also been applied to newly discovered pathways such as those involving small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi‐interacting RNAs (piRNAs). The first two pathways have been well characterized largely by in vitro methods, which need to be developed for piRNAs. Finally, new techniques, such as single‐molecule studies, are continuously being established, providing new and important insights into the field. Thus, in vitro approaches have been, are, and will continue being at the forefront of RNA research. WIREs RNA 2012, 3:509–527. doi: 10.1002/wrna.1115 This article is categorized under: RNA Processing > Splicing Mechanisms Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action RNA Methods > RNA Analyses In Vitro and In Silico

Types of in vitro systems by degree of complexity. Flowchart for the types of in vitro systems, from low (top) to high degree of purification (bottom). The cell nucleus, cytosol, and organelles are indicated in green, yellow, and gray, respectively.

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Basic assays in miRNA and siRNA pathways. (a) Dicing assays. Radiolabeled long dsRNA is added to whole‐cell or cytoplasmic extracts, and the siRNA products are observed by gel. (b) Pri‐miRNA processing (Drosha) assays. Radiolabeled, in vitro‐transcribed primary miRNA precursors are incubated with NEs or WCEs. (c) Slicing or Argonaute cleavage assays. Cell extracts, Argonaute‐containing purification fractions or immunoprecipitates, or recombinant Argonautes are incubated with single‐stranded RNAs containing the miRNA/siRNA target sites. The guide RNA may be endogenous to the cell type, or introduced by transfection. (d) Translational repression/deadenylation assays. Extracts competent for translation/deadenylation are incubated with luciferase mRNAs with engineered miRNA sites in the 3′ UTR. Translation rates are measured by the luciferase activity, whereas transcript deadenylation and stability is monitored by gel.

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An overview of eukaryotic small RNA pathways. (a) miRNAs are endogenous 21–25 nt small RNAs that associate with Argonaute‐family proteins and guide them to target mRNAs to downregulate their stability or translation rates (in animals), or cleave the mRNA directly (in plants). In the former case, the miRNAs are only partially complementary to the targets, while the cleavage activity requires full complementarity. miRNAs are processed by Drosha from longer primary transcripts (pri‐miRNAs) that fold into hairpin structures, releasing a shorter precursor miRNA hairpin (pre‐miRNA). A cleavage by Dicer follows to produce a double‐stranded miRNA intermediate, one strand of which is preferentially loaded into Argonaute. (b) The related siRNA pathway initiates with long dsRNA from endogenous convergent transcripts or gene–pseudogene pairs, viral infection, or artificial introduction. This trigger is processed directly by Dicer, unwound and loaded into Argonaute proteins to cleave perfectly complementary target RNAs. (c) In the piRNA pathway, 25–30 nt RNAs arising from long single‐stranded primary transcripts by largely unknown mechanisms associate with Piwi‐clade Argonaute proteins to participate in the control of transposons in the germline. In a ‘ping‐pong’ amplification loop, transcript recognition and cleavage by this complex leads to the generation of a secondary piRNA, which in turn can cleave an opposite‐strand transcript to produce more of the original piRNA.

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Basic assays on in vitro pre‐mRNA 3′‐end formation. (a) Coupled transcription and 3′‐end processing. DNA added to the extract is transcribed and 3′‐end processing occurs co‐transcriptionally, mediated by the CTD of RNA pol II (in yellow). (b) Cleavage and polyadenylation assays probe for the end‐product (mRNA with poly(A) tail) on in vitro transcribed substrates comprising the cis‐elements necessary for 3′‐end formation. (c) Cleavage assays are performed using extracts or purified components and EDTA blocking polyadenylation. (d) Polyadenylation assays are based on the addition of a poly(A) tail on pre‐mRNAs terminated near the cleavage site, mimicking a precleaved substrate.

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Pre‐mRNA processing. Schematic representation of the four processing steps occurring at the human ApoB pre‐mRNA. Gray cylinders depict exons, wavy lines introns or the 3′ end released by cleavage, and thin lines the splicing events joining the exons. The edited codon, the canonical terminal intronic dinucleotides, and the poly(A) signal are indicated, with the edited nucleotide highlighted in red. The two steps of splicing and 3′‐end formation are shown schematically. In splicing, a free upstream exon and lariat‐downstream exon intermediates are formed in the first step, and the exons are joined and the intron is excised as a lariat in the second step. In 3′‐end processing, the pre‐mRNA is cleaved 10–30 nucleotides downstream of the poly(A) signal in the first step, and addition of the poly(A) tail occurs in the second step.

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Standard in vitro pre‐mRNA splicing assays. (a) Classical in vitro splicing assay. Large volumes of suspension HeLa cells are used to prepare NE or cytoplasmic S100 extract, which is competent for splicing upon complementation. The splicing substrate generated by in vitro transcription and radiolabeled, the intermediates and products are shown schematically as in Figure 2. The migration of the branched molecules substantially varies depending on the substrate and gel percentage. Splicing for each substrate can be optimized by titrating the amount of NE, concentration of MgCl2, or addition of the crowding agent polyvinyl alcohol (PVA). (b) Spliceosome‐assembly assay. Spliceosomes are separated electrophoretically in agarose and/or acrylamide gels with (or without) the addition of heparin to prevent nonspecific aggregates. Without ATP, a nonspecific complex H (composed of hnRNPs, in yellow) assembles on the pre‐mRNA, and subsequently the E complex forms only in the absence of heparin, including U1 snRNP bound to the 5′ splice site and U2AF heterodimer (light blue) to the polypyrimidine tract. Upon ATP addition, a prespliceosome A complex is formed in which the U2 snRNP binds the branch‐point sequence. The other three snRNPs (U4, U5, and U6) join the complex to form the precatalytic spliceosome B, and subsequently U1 and U4 snRNPs and U2AF leave the spliceosome to form the catalytic C (as well as the B*) complex. These are simplified descriptions of the complexes, as some factors (like the Prp19 complex) are not shown. (c) Coupled transcription‐splicing assay. Template DNA incubated in NE is transcribed, and co‐transcriptional splicing occurs as introns are excised before transcript release from RNA pol II.

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Pre‐mRNA editing. (a) Cytosine‐to‐adenosine deamination. S100 extracts from vertebrate liver or intestinal cells or a reconstituted editosome including APOBEC and ACF are capable of editing pre‐mRNAs harboring the cis‐acting elements, including the mooring sequence (M) a few nucleotides downstream of the edited cytosine. (b) Adenosine‐to‐inosine deamination. Recombinant ADAR1 or ADAR2 incubated with natural‐target or synthetic pre‐mRNAs catalyze either site‐selective or promiscuous adenosine deamination. Substrates are mostly double‐stranded and include mismatches, loops, and bulges, which influence the degree of editing. Inosines are detected and quantified by hybridization, primer extension, cloning and sequencing, or mononucleotide chromatography.

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Pre‐mRNA capping/decapping. Schematic representation of the capping and decapping steps with the corresponding enzymes characterized in vitro. Early after transcription initiation by pol II (promoter indicated by arrow), the nascent pre‐mRNA is capped by a three‐step reaction: (i) RNA‐5′ triphosphatase‐mediated hydrolysis to generate 5′‐diphosphate pre‐mRNA; (ii) addition of GMP to the 5′ end of the diphosphate pre‐mRNA forming a 5′ → 5′ bond by guanylyltransferase; and (iii) addition of N‐7 methyl group by a specific methyltransferase, which can modulate the activity of the cap. The substrates and products for each reaction are shown. S‐Adomet and S‐Adohcy correspond to S‐adenosylmethionine and S‐adenosylhomocysteine, respectively. The triphosphatase and guanylyltransferase activities are carried out by separate proteins in yeast but by a single enzyme in humans. Upon completion of capping, transcription proceeds and the subsequent steps in gene expression take place. In the cytoplasm, mRNAs are repressed by decapping (removal of the cap leaving a 5′ monophosphate) by the Dcp2 enzyme and its cofactor Dcp1.

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RNA Methods > RNA Analyses In Vitro and In Silico
RNA Processing > Splicing Mechanisms
Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action

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