Hi Sue,

You have a few options here, some of which will depend on knowing a bit more about the protein you're interested in.  Here are a few ideas:

1) If you think it's a fairly broad-specificity RNA binding protein, or you have some reason to suspect it binding a particular region/sequence of RNA, you can do a basic in vitro bandshift assay - label an RNA with radioactivity/fluorescence (you can try various ones - oligoA, oligoC, gene sequence, hairpins....), incubate it with increasing concentrations of purified protein and run it out on a native gel, looking for increased migration of the RNA up the gel (i.e. at a higher molecular weight - indicative of binding of your protein) at higher concentrations.

2) If you suspect particular RNAs of binding to your protein, you could take a RIP or a CLIP approach - RNA immunoprecipitation or Cross-link IP - and do an RT-PCR or Northern to check for them. 

3) If you suspect your protein of binding to mRNA but don't know which ones, you can potentially do a nonspecific poly(A) Northern approach - do a RIP as above, run them out on a gel and probe with radio- or fluorescently-labeled oligo(dT) to non-specifically detect polyadenylated RNAs.  If you suspect other RNA species (rRNA, sno/snRNA, tRNAs) you may also be able to design probes for these, but you'd have to have more specific information.  If you're looking at lncRNAs/cRNAs or similar, it might be a problem.

4) If you're really not sure what kind of RNAs you're binding to, you can always take a completely unbiased approach and start looking at CLIP-Chip or CLIP-Seq methods to identify by high-throughput methods all the RNAs bound to your protein by microarray/HT sequencing; if you generate data from this, there's a number of algorithms out there to help with motif enrichment analysis etc. which may help guide your subsequent bioinformatic analyses.  Be aware that these are quite complicated/expensive methods, however, and you probably want to have some other evidence for definite RNA binding first.

5) To take an entirely different approach, if you have a phenotype arising from knockdown/deletion of your protein, you could always generate domain-specific deletion or point mutants and see if loss of the putative RNABD are able to replicate these phenotypes?  It's less specific, but may be a starting point.

I hope all that is of some use - I'm aware it's qujite hand-wavy at the moment, but without knowing more about your protein it's hard to be more concrete.  One thought - if you know the particular type of RNABD in your protein (ZnF, RRB, WDR), you may be able to use some bioinformatic structure prediction/RNA binding programmes online to give you some guidance on possible sequence/type of RNA bound, or alternatively compare your domain to other known structures of that domain type using, e.g., i-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) to give you a little more guidance on what kind of sequences/structures to look at.

Hope that helps!

Dan

Dear Sue,

The answer to your question can be found in the following text:

Methods for Detecting Protein-RNA Interactions

Protein Biology Resource Library

Pierce Protein Methods

Proteins interact with RNA through electrostatic interactions, hydrogen bonding, hydrophobic interactions and base stacking in a manner similar to protein-DNA interactions. Protein-RNA interactions are also significantly influence by the tertiary structure on the RNA molecules. Therefore, in assays to identify protein-RNA interactions, both the RNA and protein(s) must be correctly folded to allow proper binding. RNA is very susceptible to degradation, so special care must be taken not to introduce RNases into the reaction. Protein-RNA interactions are required to both transport a messenger RNA molecule into the cytoplasm of a eukaryotic cells and for formation of the translation machinery. The rate of translation and stability of RNA are also influenced by protein-RNA interactions as well as RNA-RNA interactions. The most common methods for studying protein-RNA interactions are discussed below.

RNA Electrophoretic Mobility Shift Assay

The RNA-EMSA is an in vitro technique used to detect protein-RNA interactions through changes in migration speed during gel electrophoresis. First, a labeled RNA probe is incubated with a protein sample (typically from a cell lysate) to initiate binding and formation of the interaction complex. The binding reaction is then separated via non-denaturing polyacrylamide gel electrophoresis. Like protein-DNA complexes, a protein-RNA complex migrates more slowly than a free RNA probe through a gel matrix. This causes a migration shift relative to the nonbound RNA probe. Specificity is determined through a competition reaction, where excess unlabeled RNA is incubated in the binding reaction, resulting in a decrease in the shifted signal if the labeled and unlabeled RNA sequences compete for binding of the same protein. Alternatively, the protein-RNA complex may be crosslinked and the reaction run on a denaturing gel. Specificity is determined through visualization of a single shifted band. Traditionally, RNA probes are radioactively labeled for detection, although fluorescent and chemiluminescent detection is also possible. Non-radioactive RNA end-labeling techniques are limited, but more versatile biotin and fluorescent labeling methods are now available.

Strengths

Limitations

non-radioactive detection available

easy to screen RNA mutants for binding efficiency

compatible with RNA labeled via run

off transcription reactions, but best results from end-labeled RNA probes

homebrew experiments require significant reagent optimization for success

lengthy protocol including native electrophoresis step

antibodies needed to determine identity of RNA binding protein

requires labeled RNA probe design and synthesis

Learn more

Methods for Labeling Nucleic Acids

View Products

LightShift Chemiluminescent RNA-EMSA Kit

Pierce RNA 3' End Biotinylation Kit

Protein Interactions Handbook

Our 72-page Protein Interaction Technical Handbook provides protocols and technical and product information to help maximize results for Protein Interaction studies. The handbook provides background, helpful hints and troubleshooting advice for immunoprecipitation and co-immunoprecipitation assays, pull-down assays, Far-Western blotting and crosslinking. The handbook also features an expanded section on method to study protein:nucleic acid interactions, including ChIP, EMSA and RNA EMSA. The handbook is an essential resource for any laboratory studying Protein Interactions.

Contents include: Introduction to Protein Interactions, Co-Immunoprecipitation, Pull-Down Assays, Crosslinking Reagents, Label Transfer, Far-Western Blotting, Protein Interaction Mapping, Yeast Two-hybrid Reporter Assay, Electrophoretic Mobility Shift Assays [EMSA], Chromatin Immunoprecipitation Assays (ChIP), Protein:Nucleic Acid Conjugates, and more.

  Protein Interactions Handbook

RNA Pull-down Assay

RNA Pull-down Assays selectively extract a Protein-RNA complex from a sample. Typically, the RNA pull-down assay takes advantage of high affinity tags, such as biotin or azido-phosphine chemistry. RNA probes can be biotinylated, complexed with a protein from a cell lysate and then purified using agarose or magnetic beads. Alternatively, the protein may be labeled, or the RNA-Protein complex may be isolated using an antibody against the protein of interest. The RNA is then detected by Northern blot or through RT-PCR analysis and the proteins detected by Western blotting or mass spectrometry.

Strengths

Limitations

enrichment of low abundant targets

isolation of intact complexes

compatible with immunoblotting and mass

spectrometry analysis

RNA secondary structure is important for function,

so end-labeling is preferred

inefficient elution from the resin

nuclease-free conditions with the beads/resin

Learn more

Methods for Labeling Nucleic Acids

View products

Pierce Magnetic RNA-Protein Pull-Down Kit

Pierce RNA 3' End Biotinylation Kit

Oligonucleotide-Targeted RNase H Protection Assays

RNase protection assays (RPA) are a powerful method for detecting RNA and RNA fragments in cell extracts. Unlike Northern blotting or RT-PCR analysis, RPA assays allow greater flexibility in the integrity of target RNA, requiring very short segments for hybridization and detection. RPA assays can also be used to map protein-RNA interactions. In this adaptation of the RPA, RNase H is used to cleave a target RNA molecule at a specific site hybridized with a DNA probe. If a protein is bound to the RNA at the target sequence, it will prevent will block probe hybridization, prevent cleavage by RNase H and indicate a site of interaction between protein and RNA. RNase H requires only a four basepair hybrid with a DNA probe in order to cleave the RNA molecule of interest. Using many small probes allows the entire sequence of RNA to be mapped for sites sites of interaction.

Strengths

Limitations

can be use for in vitro assays and crude cell extracts

allows detailed mapping of protein-RNA interactions

can be used to for functional and mutational studies

many probes required for detailed mapping

difficult to optimize for rare RNA molecules

not compatible with high throughput

Learn more

Methods for Detecting Protein-DNA Interactions

Fluorescent in situ Hybridiation Co-localization

Fluorescent in situ Hybridization (FISH/ISH) co-localization techniques require detection of both a RNA transcript and a protein of interest using RNA probes and antibodies. FISH/ISH detects the position and abundance of a RNA and protein in a cell or tissue sample. The read-out is visual (usually imaged via microscopy) and a co-localized signal for both the RNA and protein of interest indicate possible complex formation. A labeled RNA probe must be generated for detection of a particular sequence of RNA and the protein may be detected using antibody staining or fluorescent protein constructs.

Strengths

Limitations

provides a snap-shot of RNA-Protein interactions as they occur in vivo

possible to multiplex with different substrates

delivers publication quality images

tedious procedure, requiring denaturation, hybridization and detection

detection usually requires signal amplification

cells/tissue must be preserved

quantitative software is necessary to determine co-localization

Learn more

Methods for Detecting Protein-DNA Interactions

References

Selected References for Studying Protein-RNA Interactions:

Friedman, R. C., et al. (2009). Most mammalian mRNAs are conserved targets of microRNAs. Genome Research 19: 92-105.

Cannell, I. G., et al. (2008). How do microRNAs regulate gene expression? Biochem. Soc. Trans. 36: 1224-1231.

Mendes, N. D., et al. (2009). Current tools for the identification of miRNA genes and their targets. Nucleic Acids Research 37: 2419-2433.

Eulalio, A., et al. (2008). Getting to the root of miRNA-mediated gene silencing. Cell 132: 9-14.

Datta, J., et al. (2008). Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res 68: 5049-5058.

Meister, G. (2007). miRNAs get an early start on translational silencing. Cell 131: 25- 28.

Su, H., et al. (2009). Essential and overlapping functions for mammalian Argonautes in microRNA silencing. Genes & Dev. 23: 304-317.

Farazi, T. A., et al. (2008). The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 135: 1201- 1214.

Tu, K., et al. (2009). Combinatorial network of primary and secondary microRNA-driven regulatory mechanisms. Nucleic Acids Res. 37: 5969-5980.

Pesole, G., et al. (2002). UTRdb and UTRsite: specialized databases of sequences and functional elements of 5' and 3' untranslated regions of eukaryotic mRNAs. Update 2002 Nucl. Acids Res. 30: 335-40.

Pesole, G., et al. (1997). Structural and compositional features of untranslated regions of eukaryotic mRNAs.Gene 205: 95-102.

Klausner, R.D., et al. (1993). Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72: 19-28.

Ying, B., Fourmy, D., and Yoshizawa, S. (2007). Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA. RNA 13: 2042-2050.

Gunzl, Arthur and Bindereif A (1999). Oligonucleotide-targeted RNase H protection analysis of RNA-protein complexes. Methods Mol Biol. 118:93-103.

Hoping this will be helpful,

Rafik

You can use RNA-immunoprecipitation to check whether the protein is interacting with the RNA. If the interaction is of low affinity you can use CLIP (cross-linked and immunoprecipitation). Also you can use REMSA (RNA electromobility shift assay) or direct in vitro cross-linking assay (including RNase treatment). There are also some techniques such as SPR (surface plasmon resonance), ITC (Isothermal titration calorimetry) and Fluorescence Anisotropy which you can use to validate your RNA-protein interaction.

There are various of method to test RNA binding capability of a protein. If you can obtain the recombinant and purified protein, you can easily try EMSA to have some idea first. More quantitatively, you can employ fluorescence anisotropy assay to measure the Kd. If grant is not a problem, Biacore is a good option. By synthesize biotin-labelled RNA, you can immobolize the probe to chip and flow you protein to measure the binding affinity. Addtionally, ITC is also a good choice. 

Hi Sue,

I have successfully used this method to validate the RNA-binding potential of several proteins:

Strein C et al., A versatile assay for RNA-binding proteins in living cells, RNA. 2014 May;20(5):721-31 (http://www.ncbi.nlm.nih.gov/pubmed/24664470)

Basically, you express a GFP version of your protein, cross link your cells with UV, IP the GFP, hybridize a fluorescent (red) oligo-dT to the RNA, and quantify both green and red fluorescence. With an appropriate control, the red/green ratio will tell you if your protein is an RNA binding protein... (It's only suitable for polyadenylated RNA...)

Good luck

http://www.ncbi.nlm.nih.gov/pubmed/24664470

Hi Sue,

You may use Electrophoretic Mobility Shift Assay (EMSA) , a powerful method to study the protein-DNA interaction, is adapted for RNA-protien complexes. You may find its details from this paper: http://cshprotocols.cshlp.org/content/2014/4/pdb.prot080721

Good luck,

Thank you for all the answers. They are really helpful.

Dear all,

I am looking for the information about RNA and protein binding assay on the contrary direction. In my case, the RNA is knowm, I'd like to check if it is possible to bind any un known proteins.  I could not use EMSA to do because I don't which which protein is binding by this RNA.

In addation, this RNA has tRNA-like structure. Do you think it may have the function as tRNA?

thanks,

Hello dear colleague,

I think the RNase protection assay is a convenient test for such study. Its requirements are just treatment of mixture of protein-of-interest and your RNA with RNase and cDNA synthesis followed by PCR.

Good luck.

Mingmin Zhao In your case you can in vitro transcribed your RNA and body label it with biotinylted Uracil in the transcription step or You can end label your RNA with Biotin. After that bind your labelled RNA with Magnetic streptavidin beads followed by incubation with whole protein pool. Collect the beads and wash it several times with neutral pH Tris and elute with 1X SLD. Run in gel , perform colloidal comassie or silver stain. Any differential band if you get , mass spec the spot and identify the binding protein. If further details required, you can ping me in my id [email protected]

Sue yu If your protein is known You can perform RIP and perform PCR using primers against any predicted RNA.

Or entire pulled down RNA , you can send for RNA Seq and identify the binding RNA to your protein. Hope it will help.

There are several methods that can be used to test whether a protein can bind to RNA directly, even if the potential RNA targets are unknown. Some commonly used techniques include:

It's worth noting that some of these methods may require optimization or modifications to work well with your specific protein of interest. And some methods like RNA-SELEX may take time and lots of resources.