RNA-protein interactions are key to understanding human health and disease. Crosslinking-immunoprecipitation (CLIP) and related technologies are powerful tools for characterizing these interactions. Since their introduction in 2008 (Licatalosi et al., 2008), CLIP-based approaches have been applied to prominent RNA research fields such as HIV and cancer.
Recently, Sebla Kutluay and colleagues used CLIP to characterize the global changes in the RNA binding of HIV viral proteins (Kutluay et al., 2014). They found that specifically during viron genesis, HIV protein Gag binding specificity changes facilitating genome packaging. They also showed Gag binds to specific host tRNAs, not viral RNA.
Similarly, Yoon et al. recently presented comprehensive data on the cancer and aging-related protein AUF1. They found that it regulates RNAs by lowering or enhancing steady-state levels, and promoting translation (Yoon et al., 2014).
These and numerous other recent findings have been made possible by various CLIP technologies suited for each investigation. Let’s take a look at some of the most widely used methods today.
Breaking Down the CLIP Approaches
CLIP protocols all involve RNA-protein cross-linking followed by immunoprecipitation against a protein of interest. Ultraviolet light is used to create cross-links between RNA and protein in vivo (Brimacombe et al., 1988). The RNA is then isolated and reverse transcribed into cDNA, which can then be used on a host of platforms to identify and quantify interacting RNAs. Several refinements and specializations of this central CLIP principle exist: CLIP-seq, PAR CLIP and iCLIP are three of the most common.
CLIP followed by sequencing (CLIP-seq) identifies all RNA species bound by a protein of interest (Licatalosi et al., 2008). After UV cross-linking and immunoprecipitation, RNA is purified by proteinase digestion. A cDNA library is then created from the RNA. This DNA library is compatible with most next-generation sequencing platforms. After sequencing, the identity and amount of the interacting RNAs is obtained.
Pros: Adaptability and relative ease of sample preparation have maintained CLIP-seq as a principal technology in RNA research. For example, a common application of CLIP-seq is generation of detailed splice maps (Konig et al., 2010; Xue et al., 2009).
Cons: UV light may cause mutations in DNA and has low efficiency compared with formaldehyde crosslinking. This means that CLIP-seq is not as precise or high-resolution as other methods.
Popular use: Researchers choose CLIP-seq when broad genome coverage, and a balance of resolution and experimental ease are necessary.
Additional Resource for CLIP-seq
HITS-CLIP: panoramic views of protein-RNA regulation in living cells: This review provides more background on CLIP-seq, its uses and limitations.
PAR CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) is a further innovation based on CLIP-seq. It involves incorporation of photoreactive ribonucleoside analogs into the RNA of living cells.
These analogs readily and specifically cross-link to interacting proteins under UV light. Interactions are isolated and the protein linkages are removed allowing RNA purification and reverse transcription, while mutations are left by the linkages at the interaction points. After sequencing, these characteristic mutations are easily identified against the reference sequence, allowing single-nucleotide resolution of binding events (Spitzer et al., 2014).
Pros: This system has increased resolution and decreased signal-to-noise ratio over CLIP-seq.
Cons: Requires an experimental system amenable to RNA alteration, such as single-celled organisms and cell-culture.
Popular Use: PAR CLIP is well suited to study basic post-transcriptional processes in cell culture, and is the gold standard in this field.
Additional Resource for PAR CLIP
Identification of RNA-protein interaction networks using PAR-CLIP: This review provides an in-depth discussion of the crosslinking methods that set PAR-CLIP apart from other CLIP methods, while also introducing the pros and cons of this type of approach.
iCLIP (individual-nucleotide resolution CLIP) uses a 3’ exonuclease to degrade protein-bound RNA. This enzyme digests the isolated RNA but stops at the cross-linked protein. An adapter is then ligated to this position. After reverse transcription and sequencing, the presence of this adapter in the sequence immediately follows the exact binding site in the RNA. (Huppertz et al., 2014).
Pros: iCLIP does not require cells in culture and can be used in most experimental systems.
Cons: iCLIP employs a more meticulous protocol with a great deal of bioinformatic analysis.
Popular Use: Researchers choose iCLIP when resolution is paramount, and it can be used when PAR CLIP is not possible (i.e. in animal tissue).
Additional Resources for iCLIP
Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions: This review provides a thorough commentary of the experimental differences with the iCLIP protocol and the unique advantages it offers. It also provides a comparative look between the experimental results from the CLIP and iCLIP approaches.
iCLIP protocol download: An optimized protocol for iCLIP can be downloaded directly from our website.
A CLIP for Every Occasion
Together, these CLIP technologies form a toolbox for understanding RNA biology at the finest resolution and broadest scale. Like many applications, there isn’t really a “best” approach, but rather one that is most appropriate, depending on your experimental goals and unique research scenario.
Antibodies for Use in CLIP Methods
When choosing an antibody for use in any of the CLIP-based methods, we suggest starting with well-characterized antibodies that have been demonstrated to work in some applications that involve immunoprecipitation, such as a standard IP or chromatin immunoprecipitation (ChIP). Find Abcam antibodies known to work in CLIP here.
- Brimacombe R, Stiege W, Kyriatsoulis A, Maly P (1988). Intra-RNA and RNA-protein cross-linking techniques in Escherichia coli ribosomes. Methods Enzymol. 164, 287–309.
- Huppertz I, Attig J, D’Ambrogio A, Easton LE, Sibley CR, Sugimoto Y, Tajnik M, Konig J, Ule J (2014). iCLIP: Protein-RNA interactions at nucleotide resolution. Methods 65, 274–287.
- Konig J, Zarnack K, Rot G, Curk T, Kayikci M, Zupan B, Turner DJ, Luscombe NM, Ule J (2010). iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915.
- Kutluay SB, Zang T, Blanco-Melo D, Powell C, Jannain D, Errando M, Bieniasz PD (2014). Global changes in the RNA binding specificity of HIV-1 Gag regulate virion genesis. Cell 159, 1096–1109.
- Licatalosi DD, Mele A, Fak JJ, Ule, J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (2008). HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469.
- Spitzer J, Hafner M, Landthaler M, Ascano M, Farazi T, Wardle G, Nusbaum J, Khorshid M, Burger L, Zavolan M, Tuschl T (2014). PAR-CLIP (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of RNA-binding proteins. Methods Enzymol. 539, 113–161.
- Xue Y, Zhou Y, Wu T, Zhu T, Ji X, Kwon YS, Zhang C, Yeo G, Black DL, Sun H, Fu XD, Zhang Y (2009). Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping. Mol. Cell 36, 996–1006.
- Yoon JH, De S, Srikantan S, Abdelmohsen K, Grammatikakis I, Kim J, Kim KM, Noh JH, White EJ, Martindale JL, et al. (2014). PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity. Nat. Commun. 5, 5248.