As geneticist Theodosius Dobzhansky said1 in 1973, “Nothing in biology makes sense, except in the light of evolution.” Many modern biologists may add that nothing in molecular biology makes sense, except in the light of biochemistry – without the quantitative understanding that biochemistry provides, as biologists can predict the effect of a double reduction in the levels of a protein during the initial development of a protein. organism, or a tenfold increase in the concentration of another protein in cancer cells? The chasm between simplified biochemistry experiments and the complicated complexity of the cell has long since seemed insurmountable. Now, writing in Nature, Sharma et al.two report a technique that allows biochemical analysis of molecular interactions in cells.
The authors focused on the dynamics of interactions between RNA molecules and proteins. The messenger RNA molecules are linked by various RNA binding proteins (RBPs), which control almost every aspect of the mRNA life cycle – from the initial processing of newly produced RNAs to their eventual destruction3. Each RBP can bind to hundreds of RNA molecules and, in turn, each RNA can be linked by dozens of different RBPs4. In addition, RNA-protein interactions are not static5,6. Instead, proteins can quickly bind to their target RNAs and, just as quickly, dissociate from them (Fig. 1), and these dynamics are at the heart of gene regulation. In other words, the kinetics of RNA-protein interactions are a driving force for gene expression. Defining the parameters of these kinetics in cells is, therefore, crucial for a complete understanding of the regulation of gene expression.
Figure 1 | A method for probing RNA-protein interactions in cells. Proteins that act on RNA molecules quickly associate and dissociate from their target binding sites. Measurements of association and dissociation rates are necessary for a quantitative understanding of gene regulation, but have been impossible to do in living cells. Sharma et al.two describe a method called KIN-CLIP that uses ultrafast pulses of ultraviolet light to generate covalent cross-links between bound proteins and RNA molecules in cells. This not only allows the RNA targets of proteins to be identified (as was possible in crosslinking techniques previously reported), but, due to the speed of the crosslinking process, it also allows the association and dissociation kinetics to be determined.
Although RNA-protein interactions have been investigated for decades, their kinetics in cells have not been characterized. In general terms, kinetic insight is only available in in vitro studies using purified proteins; Cell experiments have been able to identify RBP RNA targets, but lacked the precision to measure the kinetics of interactions5. With the advent of high-throughput sequencing methods, in vitro approaches can now probe the kinetics of a protein’s interactions with tens of thousands of RNA variants7. But these experiments are still carried out on purified proteins in the absence of the cellular medium. In recent years, a method called cross-linking and immunoprecipitation8 (CLIP) has become a workhorse for the characterization of RNA-protein interactions in cells. In CLIP, a protein in complex with an RNA molecule is covalently cross-linked to RNA using ultraviolet light; the complexes are then isolated and the crosslinked RNA is identified by high throughput sequencing. This approach provides a catalog of RNAs that bind to a specific RBP in the complex cell environment, but it provides, at best, only a snapshot of these interactions.
Sharma and colleagues now bridge the gap between in vitro strategies and CLIP through the development of a type of CLIP that can determine the kinetic parameters of RNA-protein interactions in cells. The authors’ main insight was that certain technical aspects of the CLIP methods previously reported prevented such approaches from being useful for capturing kinetic parameters. The most challenging limitation is that crosslinking rates must be fast to capture the rates at which proteins and RNA molecules associate and dissociate. Conventional UV sources cannot achieve crosslinking fast enough, so using them to measure kinetics is like using a slow shutter speed to photograph a galloping horse – everything blurs in the image. This finding led the authors to use a pulsed femtosecond ultraviolet laser, which cross-links proteins to RNA fast enough to capture kinetic parameters. They call their method KIN-CLIP (for kinetic CLIP).
To test the method, the authors applied it to an RBP called Dazl, which is necessary for the production of reproductive cells and regulates gene expression9. Dazl binds to hundreds of target mRNAs, increasing its stability and the number of proteins produced10. However, despite its biological importance, much about Dazl’s binding and function is unknown, making him an ideal candidate for KIN-CLIP experiments.
Sharma and co-workers first found that KIN-CLIP identifies RNA targets found in previously published data sets produced from the ‘snapshot’ CLIP. They then calculated kinetic parameters, known as rate constants, for the association and dissociation of Dazl with each of its thousands of binding sites in RNA. These results revealed that Dazl’s bonding is highly dynamic: his bonding time is short; the RBP resides on individual sites for just a few seconds. Dazl also binds rarely, so the binding sites are free of the protein most of the time.
The authors also found that several Dazl molecules tend to bind in close proximity. Kinetic analysis suggests that this may be due to cooperative binding – a phenomenon in which the binding of one protein to a location increases the likelihood that other proteins will bind to nearby locations. Finally, the authors incorporated the recently determined Dazl kinetic parameters into a predictive model of their impact on gene expression, thus providing a biochemical basis for their function and setting the stage for future research.
One of the most interesting aspects of this study is the potential of KIN-CLIP to study other RBPs, but the method has some limitations. For example, as with all CLIP-based techniques, the ability to cross-link the protein of interest to bound RNAs is a requirement; this can be challenging, because some proteins do not have the necessary side chains properly oriented for crosslinking. The biggest obstacle, however, for potential KIN-CLIP converters is that specialized crosslinking equipment is required: pulsed femtosecond lasers may not be easily accessible to many biologists. In addition, the experimental procedures and associated analysis of KIN-CLIP libraries are more complicated than those of standard CLIP experiments and can be another barrier to adoption.
However, this study brought the tools of biochemistry to living cells and, in doing so, can provide an inflection point in the study of RNA-protein interactions. The next step is to apply KIN-CLIP to other RBPs, but the prospect of applying it to other types of interacting biomolecules also shines on the horizon. In fact, the authors note intriguingly that femtosecond pulsed lasers can crosslink proteins to DNA – perhaps a ‘DNA KIN-CLIP’ is within reach. Sharma and colleagues not only set a new standard in RNA biology, they may also have unleashed the power of biochemistry in molecular biology more generally.