Amber Dance is an award-winning freelance science writer based in Southern California. She is the ALS (Lou Gehrig’s disease) reporter for the Alzheimer Research Forum. She contributes to The Scientist and Nature journals, and has written about topics ranging from record-breaking rocks to bizarre new ant species.
The first step in making a protein is transcription of the code from DNA, but that’s just the beginning of a complex, tightly regulated process. A host of proteins then edit the RNA, shepherd it from the nucleus to the cytoplasm, control its stability and direct its translation. Hundreds of RNA-binding proteins (RBPs) make up that host, says associate professor Sarath Chandra Janga of Indiana University at Indianapolis. Janga, who maintains a database of human RNA-binding proteins, estimates some 3,000 or so human proteins attach to RNA. He adds that 1,400 of those have been confirmed experimentally [1,2,3].
That fact is keeping many scientists busy identifying which RNAs bind to which proteins.
Co-immunoprecipitation is the experimental method of choice, with either RNA or RNA-binding proteins as the bait, but there are alternative options for researchers.
The main choice is between ribonucleoprotein immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP). Both use antibodies to an RNA-binding protein of interest (or antibodies to an epitope tag attached to that protein) to pull down attached RNAs, but with slightly different protocols. “They both have their uses, and they both have their shortcomings,” says Jack D. Keene, professor of molecular genetics and microbiology and James B. Duke professor in medicine at Duke University Medical Center, who invented the original methods in the 1990s.
RIP vs. CLIP
To perform a RIP assay, scientists pull down the protein-RNA complex from a cell extract, then use sequencing—either high-throughput techniques or microarray-based sequencing—to identify those target RNAs. Keene prefers this technique, because it brings down everything that’s naturally attached to the RBP of interest. It’s also quantitative: The data tells scientists if a certain RNA-binding protein attached to tens, hundreds or thousands of a given RNA.
But what the classical RIP assay won’t tell is which specific five to 10 nucleotides are at the binding site for that protein; it just identifies a set of targeted RNAs. Plus, it doesn’t pull out only the RNA bound to the protein of interest, Janga says. Many RBPs often associate together in a ribonucleoprotein complex, and RIP may bring down all those associated proteins, along with any other RNAs they’re bound to at the time. In other words, a RIP assay with an antibody for RBP A could also collect RNAs that are bound to RBPs B, C and so on, in the same complex.
CLIP aims to get around those issues, limiting the IP to only the RBP of interest and the snippets of RNA it’s interacting with. Before immunoprecipitating, scientists use UV light to tightly crosslink the binding partners. Then, after the IP step, they use enzymes to shear away all RNA and proteins not protected by the antibody. As with RIP, scientists follow that step with sequencing to identify the pulled-down RNAs. Because most of the uncrosslinked RNA is removed, CLIP can identify the specific sequences where the RBP sat down.
However, CLIP also has its issues. Crucially, the UV does not crosslink nearly all the RNA-protein interactions in cells.
Scientists must often perform trial-and-error experiments to determine the amount of UV exposure that crosslinks true, important biological interactions and avoids other RNA-protein combinations that can occur but may have little functional relevance.
“The [crosslinking] efficiencies can be as low as 5% or less,” says Keene. “You’re missing a significant fraction of the binding sites.” For that reason, CLIP isn’t quantitative like RIP is—it can’t distinguish the relative affinities of a given RBP for one or another RNA target.
Scientists have come up with multiple versions of CLIP to improve results [4]. Keene thinks he’s addressed many shortcomings of previous methods with a new technique, digestion optimized RIP (DO-RIP), which combines the best of RIP and CLIP. By adding a partial nucleotide-digestion step to fragment protein-bound RNA to about 20 to 70 nucleotides before immunoprecipitation, DO-RIP can both quantify the amount of target an RBP binds and identify the specific binding site on the RNA, Keene says. The procedure also uses a parallel IP with nonspecific antibodies, such as plain serum, as a negative control to quantify background signal [5,6]. “The RIP now gives more coverage of the transcriptome, precision of binding events and biological outcomes,” Keene says.
Helpful products
Few companies have developed kits for RIP or CLIP, but Keene assigned his RIP and CLIP patents to MBL International Corporation. It offers a RIP-Assay Kit, including buffers optimized for RIP and rabbit IgG to use as a negative control.
Regardless of the specific technique used, antibody quality is key. MBL has about 120 antibodies to RNA-binding proteins, and of those, they’ve carefully validated about 50 as effective for RIP. The independent ENCODE project lists 1,027 antibodies for RBPs in human and mouse, of which it has so far said 224 meet its standards for binding target proteins.
MBL also offers a kit to go in the other direction: starting with the RNA of interest and identifying bound proteins. To use the RiboTrap Kit, researchers first synthesize their RNA bait using 5-bromo-UTP and then incubate the synthetic RNA with a cell lysate. Then, they use the included anti-BrdU antibody to pull down that RNA, plus any associated proteins, and identify the proteins by mass spectroscopy. Users can choose from three wash buffers to find strong or weak associations between the RNA and RBP. But just because an interaction is strong doesn’t mean it serves a crucial biological function, and weak interactions could be important, cautions Shinobu Kitamura, director of regulatory affairs at MBL.
As for CLIP, only a handful of labs have really mastered it, says Janga. The data can get noisy, with many possible RNA targets popping up. It helps to perform replicates, he says—both from the same sample and from different samples treated the same way. “Doing these biological and technical replicates really could increase the power and quality of your identified binding sites,” Janga says. Keene also recommends biological replicates of DO-RIP experiments, with cell lysates collected at least three different times.
The toolbox for examining RNA-protein interactions and complexes continues to evolve. Tool providers are working with scientists to develop more specific tools, which will help researchers gain a better understanding of the role of RNP complexes. Stay tuned as scientists continue to uncover and identify specific RNAs that are interacting with various proteins in their research efforts to fine-tune their studies on the regulatory pathways in the cell.
References
[1] Zhao, H, et al., “Prediction and validation of the unexplored RNA-binding protein atlas of the human proteome,” Proteins, 82:640-7, 2014. [PMID: 24123256]
[2] Neelamraju, Y, et al., “The human RBPome: from genes and proteins to human disease,” J Proteomics, 127:61-70, 2015. [PMID: 25982388]
[3] Hashemikhabir, S, et al., “Database of RNA binding protein expression and disease dynamics (READ DB),” Database, 2015:bav072, 2015. [PMID: 26210853]
[4] Janga, SC, “From specific to global analysis of posttranscriptional regulation in eukaryotes: posttranscriptional regulatory networks,” Brief Funct Genomics, 11:505-21, 2012 [PMID: 23124862]
[5] Nicholson, CO, et al., “DO-RIP-seq to quantify RNA binding sites transcriptome-wide,” Methods, epub ahead of print, 2016. [PMID: 27840290]
[6] Nicholson, CO, et al., “Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq,” RNA, epub ahead of print, 2016. [PMID: 27742911]
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