As geneticist Theodosius Dobzhansky put it1 in 1973, “Nothing in Biology Makes Sense Except in the Light of Evolution.” Many modern biologists can add that nothing makes sense in molecular biology except in the light of biochemistry – without the quantitative understanding that biochemistry offers, how can biologists have the effect of a dual reduction in protein levels during the early development of an organism predict, or of a tenfold increase in the concentration of another protein in cancer cells? The gap between the streamlined experiments of biochemistry and the confusing complexity of the cell has long seemed insurmountable. Now, sign up Nature, Sharma et al.2 reports a technique that enables the biochemical analysis of molecular interactions in cells.
The authors focused on the dynamics of interactions between RNA molecules and proteins. Messenger RNA molecules are bound by different RNA-binding proteins (RBPs), which control almost every aspect of the mRNA life cycle – from the initial processing of newly created RNAs to their eventual destruction3. Each RBP can bind to hundreds of RNA molecules, and each RNA can in turn be bound by dozens of different RBPs4. In addition, RNA-protein interactions are not static5,6. Instead, proteins can bind rapidly to their target RNAs and secrete from them just as quickly (Fig. 1), and these dynamics are at the core of gene regulation. In other words, the kinetics of RNA-protein interactions are a driving force for gene expression. The definition of the parameters of these kinetics in cells is therefore crucial for the understanding of the regulation of gene expression.
Figure 1 | A method to investigate RNA-protein interactions in cells. Proteins that act on RNA molecules rapidly associate and distance themselves from their target binding sites. Measurements of the association rates and dissociation are necessary for a quantitative understanding of gene regulation, but it is impossible to do in living cells. Sharma et al.2 describes a method called KIN-CLIP that uses ultraviolet light pulses to generate covalent cross-links between the bound proteins and RNA molecules in cells. This not only identifies the RNA targets of the proteins (as was possible in previously reported cross-linking techniques), but due to the rapid cross-linking process, the kinetics of association and dissociation can also be determined.
Although RNA-protein interactions have been studied for decades, their kinetics in cells are not characterized. In general, kinetic insight is only available from in vitro studies using purified proteins; experiments in cells were able to identify the RNA targets of RBPs, but did not have the precision to measure the kinetics of the interactions5. With the advent of high-throughput methods, in vitro approaches can now investigate the kinetics of a protein’s interactions with tens of thousands of RNA variants7. But these experiments are still performed on purified proteins in the absence of the cellular environment. In recent years, a method called cross-linking and immunoprecipitation8 (CLIP) has become a working cell for the characterization of RNA-protein interactions in cells. In CLIP, a protein in complex with an RNA molecule is covalently linked to the RNA using ultraviolet light; the complexes are then isolated and the cross-linked RNA is identified by high throughput sequence. This approach provides a catalog of RNAs that bind to a specific RBP in the complex environment of the cell, but it provides at best only a snapshot of these interactions.
Sharma and colleagues now bridge the gap between in vitro strategies and CLIP by developing a type of CLIP that can determine the kinetic parameters of RNA-protein interactions in cells. The main insight of the authors was that certain technical aspects of previously reported CLIP methods prevented such approaches from being useful for capturing kinetic parameters. The most challenging limitation is that crosslinking must be fast to capture the rate at which proteins and RNA molecules associate and dissociate. Conventional UV sources cannot achieve sufficiently fast crosslinking, so it is like using a slow shutter speed to photograph a galloping horse, to measure it to measure kinetics. This realization led the authors to use a pulsed femtosecond UV laser, which cross-links proteins fast enough to RNA 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 needed for the production of reproductive cells, and regulates gene expression9. Dazl binds to hundreds of target mRNAs, increasing their stability and the number of proteins produced10. Despite its biological importance, however, much of the binding and function of Dazl is unknown, making it an ideal candidate for KIN-CLIP experiments.
Sharma and co-workers first verified that KIN-CLIP identifies RNA targets found in previously published datasets made from ‘snapshot’ CLIP. They then calculate 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 binding is very dynamic: its binding time is short; the RBP is located in individual places only a few seconds. Dazl also rarely bind, and so the binding sites are mostly protein free.
The authors also found that several Dazl molecules tend to bind in places that are close to each other. The kinetic analysis suggests that this may be due to cooperative binding – a phenomenon in which the binding of one protein to one site increases the likelihood that other proteins bind to nearby sites. Finally, the authors incorporated the newly established kinetic parameters of Dazl into a predictive model of its impact on gene expression, thus providing a biochemical basis for its function and determining the scene for future research.
One of the most exciting aspects of this study is the potential of KIN-CLIP to study other RBPs, but the method does have limitations. As with all CLIP-based techniques, the ability to link the protein of interest to bound RNAs is a requirement; this can be a challenge because some proteins do not have the necessary side chains that are correctly oriented for crosslinking. However, the biggest obstacle to potential KIN-CLIP converters is that specialized equipment is needed for the crosslinking: pulsating femtosecond lasers may not be easily accessible to many biologists. Furthermore, the experimental procedures and concomitant analysis of KIN-CLIP libraries are more complicated than those of standard CLIP experiments, and this may be another obstacle to adoption.
Nevertheless, this study has brought the instruments of biochemistry into living cells and may provide a turning point in the study of RNA-protein interactions. The next step is to apply KIN-CLIP to other GDPs, but the prospect of applying it to other types of interaction biomolecules is also shining on the horizon. Indeed, the authors note interestingly that pulsating femtosecond lasers can cross-link proteins to DNA – perhaps a ‘DNA KIN-CLIP’ is within reach. Sharma and colleagues not only set a new standard in RNA biology, but they also unleashed the power of biochemistry on molecular biology more generally.