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Methods for Detecting Protein-RNA Interactions

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.

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Protein Interaction
Technical Handbook

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:
  • 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
Limitations:
  • 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

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Methods for Labeling Nucleic Acids

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LightShift Chemiluminescent RNA-EMSA Kit

Pierce RNA 3' End Biotinylation Kit

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:
  • enrichment of low abundant targets
  • isolation of intact complexes
  • compatible with immunoblotting and mass spectrometry analysis
Limitations:
  • RNA secondary structure is important for function, so end-labeling is preferred
  • inefficient elution from the resin
  • nuclease-free conditions with the beads/resin

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Pierce Magnetic RNA-Protein Pull-Down Kit

Pierce RNA 3' End Biotinylation Kit

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Methods for Labeling Nucleic Acids

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:
  • 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
Limitations:
  • many probes required for detailed mapping
  • difficult to optimize for rare RNA molecules
  • not compatible with high throughput

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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:
  • provides a snap-shot of RNA-Protein interactions as they occur in vivo
  • possible to multiplex with different substrates
  • delivers publication quality images
Limitations:
  • 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

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Methods for Detecting Protein-DNA Interactions

References

Selected References for Studying Protein-RNA Interactions:
  1. Friedman, R. C., et al. (2009). Most mammalian mRNAs are conserved targets of microRNAs. Genome Research 19: 92-105.
  2. Cannell, I. G., et al. (2008). How do microRNAs regulate gene expression? Biochem. Soc. Trans. 36: 1224-1231.
  3. Mendes, N. D., et al. (2009). Current tools for the identification of miRNA genes and their targets. Nucleic Acids Research 37: 2419-2433.
  4. Eulalio, A., et al. (2008). Getting to the root of miRNA-mediated gene silencing. Cell 132: 9-14.
  5. Datta, J., et al. (2008). Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis. Cancer Res 68: 5049-5058.
  6. Meister, G. (2007). miRNAs get an early start on translational silencing. Cell 131: 25- 28.
  7. Su, H., et al. (2009). Essential and overlapping functions for mammalian Argonautes in microRNA silencing. Genes & Dev. 23: 304-317.
  8. 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.
  9. Tu, K., et al. (2009). Combinatorial network of primary and secondary microRNA-driven regulatory mechanisms. Nucleic Acids Res. 37: 5969-5980.
  10. 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.
  11. Pesole, G., et al. (1997). Structural and compositional features of untranslated regions of eukaryotic mRNAs.Gene 205: 95-102.
  12. Klausner, R.D., et al. (1993). Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72: 19-28.
  13. 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.
  14. Gunzl, Arthur and Bindereif A (1999). Oligonucleotide-targeted RNase H protection analysis of RNA-protein complexes. Methods Mol Biol. 118:93-103.

 

Written and/or reviewed by Douglas Hayworth, Ph.D.

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