Native Purification and Analysis of Long RNAs
The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation–renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2′-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.
1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA
2 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
3 Department of Chemistry, Yale University, New Haven, Connecticut, USA
4 European Molecular Biology Laboratory, Grenoble Outstation, France
Corresponding author: Marco Marcia
System-wide identification of RNA-binding proteins by interactome capture (Prot 63)
mRNA interactome capture is a novel and unbiased technique to identify the active RBPs of cultured cells. Making use of in vivo UV-crosslinking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. Following stringent washes, the mRNA interactome is determined by quantitative mass spectrometry. The protocol takes three working days for analysis of single proteins by western blot and about two weeks for the determination of complete cellular mRNA interactomes by mass spectrometry. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. Applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to define the mRNA interactome of different cell lines and to compare different biological states. [...]
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany
Corresponding author: Alfredo Castello
Cloning of small RNAs with 5’ phosphate and 3’ OH ends (Prot 40)
The following protocol describes a procedure for the purification and cloning of miRNAs and other small RNAs in the 20-30 nucleotide size range from plant tissue.
Baulcombe Lab (The Sainsbury Laboratory, Cambridge) , UK
RNA-biotin based pulldown assays for the detection of siRNA targeted genomic regions and siRNA directed histone modifications (Prot 32)
The recent discovery of RNA interference and in particular the observation that siRNAs can modulate gene expression at the level of transcription, i.e. small-interfering RNA (siRNA) directed transcriptional gene silencing (TGS) in Human cells (Matzke and Birchler 2005; Morris 2005) has illustrated the fact that RNA may be far more intricately involved in epigenetics than was previously assumed. To determine more clearly how siRNAs are interacting with the homologous genomic regions in the nucleus in human cell cultures we designed several RNA-biotin based pulldown assays which can be used alone or in combination with other known assays such as ChIP and Flag-tagged pulldown assays. Three protocols are explained in detail here. The first protocol is essentially a dual-pulldown assay employing Flag-tagged DNMT3A or antibody of choice for an endogenous protein and 5' biotin linked antisense RNA, while the second protocol is a triple pulldown assay which essentially expands upon the dual pulldown to incorporate a third pulldown which is an iteration of the ChIP and is a pulldown for H3K27me3+. The third assay described here is the biotin-RNA pulldown of a low-copy RNA that spans the siRNA targeted promoter region. Data generated from this assay is currently in submission.
The Scripps Research Institute
Department of Molecular and Experimental Medicine
The Scripps Research Institute - 10550 N. Torrey Pines Road
La Jolla, CA, 92037 , USA
RNA-chromatin immunoprecipitations (RNA-ChIP) in mammalian cells (Prot 28)
RNA-protein interactions play important roles within the cell. Using a variation of the widely-used chromatin immunoprecipitation (ChIP) assay, the potential association of cellular RNAs and candidate proteins can be evaluated in a process named “RNA-ChIP”. This technique has been successfully used in mammalian cells, for example to examine the relationship of noncoding RNAs with histone proteins or to examine interactions between viral RNAs and proteins in the host mammalian cell. In RNA-ChIP, RNA-protein interactions are fixed by reversible chemical cross-linking with formaldehyde followed by immunoprecipitation with antibodies against the candidate protein(s). RNAs that are associated with the protein are detected by reverse transcriptase-PCR (RT-PCR). The following procedure was used to examine protein-RNA interactions in mouse embryonic stem cells, but can be modified for other cell types.
Howard Hughes Medical Institute
Department of Molecular Biology, Massachusetts General Hospital
Department of Genetics, Harvard Medical School
MA, 02114, USA