A general method for rapid and nondenaturing purification of RNAs

Kieft, Jeffrey S.; Batey, Robert T.

doi:10.1261/rna.7040604

Cited by 72 publications

(80 citation statements)

References 26 publications

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“…This is observed for both the SAM-I riboswitch and the D(C209)P4-P6 domain of the group I intron (Treiber and Williamson 2001). Each RNA was purified to a single band on a denaturing gel and refolded as described (Juneau and Cech 1999;Kieft and Batey 2004;Montange and Batey 2006). Separation of the refolded RNA samples by analytical size-exclusion chromatography (SEC) clearly shows a conformational heterogeneity described by a single predominant peak preceded by a smaller peak that constituted 5-15% of the total signal (Fig.…”

Section: Limitations To Saxs Analysis Due To Refolding Artifactsmentioning

confidence: 88%

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Rambo¹,

Tainer²

2010

RNA

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Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA val , and yeast tRNA phe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

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Section: Limitations To Saxs Analysis Due To Refolding Artifactsmentioning

confidence: 88%

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Rambo¹,

Tainer²

2010

RNA

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“…A previous study used a variant hepatitis delta virus (HDV) ribozyme with imidazole-dependent activity for nondenaturing RNA purification (32). The length of the tag in this method, which includes two signal recognition particle RNAs, is quite long (∼180 nt) and the affinity between signal recognition particle RNA and the T.maritima SRP Ffh M-domain (TmaM) protein to which it binds is much lower than that of the Csy4*-RNA hairpin interaction.…”

Section: Discussionmentioning

confidence: 99%

RNA–protein analysis using a conditional CRISPR nuclease

Lee

Haurwitz

Apffel

et al. 2013

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

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RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.non-coding RNA | RNA processing | miRNA | mass spectrometry R NA molecules function together with specific binding proteins to regulate cellular pathways at the levels of transcription, posttranscriptional modification, and translation (1-4). For example, microRNAs (miRNA), siRNAs, and piwi-interacting RNAs (piRNAs) regulate more than 30% of mammalian gene expression (5). Small nucleolar RNAs (snoRNAs) govern the sites and efficiencies of RNA chemical modifications in cells (6), whereas long noncoding RNAs (lncRNAs), such as HOTAIR and MALAT1, have been implicated in chromatin remodeling, transcriptional activation, and tumorigenesis (7,8). UTRs of mRNA transcripts are also known to interact with regulatory proteins to control their expression level as well as their stability. Understanding how these RNAs function in cells and how they may be manipulated for therapeutic purposes is an important goal that spans many areas of biology.Although new ncRNAs are being discovered at a rapid pace, determination of their biochemical activities is often slow. A major barrier to such functional analysis is the current difficulty of identifying RNA-binding partners that associate with specific transcripts and participate in their biological behavior. Despite the development of various RNA affinity purification methods, the expense and/or technical challenges associated with each approach has precluded its use by nonspecialists or in high-throughput discovery experiments. Current strategies for identifying RNA-binding proteins that associate with specific transcripts involve the use of affinity tags, including biotin, aptamers, and particular proteinbinding sequences (9-13). In each case, however, the modest affinity or specificity of tag recognition and the difficulty of selective elution complicate sample analysis. A strategy that will allow simple and rapid identification of proteins that associate selectively with particu...

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“…A number of techniques have been developed for the preparation of folded RNA samples suitable for structural studies, including denaturing (Golden and Kundrot 2003;Ke and Doudna 2004) and nondenaturing methods (Kieft and Batey 2004;Batey and Kieft 2007;Kim et al 2007). …”

Section: Preparation Of Rna Samples To Minimize Refolding Artifactsmentioning

confidence: 99%

Solution structure of RNase P RNA

Kazantsev¹,

Rambo²,

Karimpour³

et al. 2011

RNA

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The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.

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A general method for rapid and nondenaturing purification of RNAs

Cited by 72 publications

References 26 publications

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Improving small-angle X-ray scattering data for structural analyses of the RNA world

RNA–protein analysis using a conditional CRISPR nuclease

Solution structure of RNase P RNA

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