Dynamics of RNA-protein interactions studied in living cells

As stated by geneticist Theodosius Dobzhansky¹ in 1973, “Nothing in biology makes sense except in the light of evolution.” Many modern biologists might add that nothing in molecular biology makes sense except in the light of biochemistry – without the quantitative understanding that biochemistry provides, how can biologists predict the effect of a double reduction in levels of a protein during the early development of an organism? , or a tenfold increase in the concentration of another protein in cancer cells? The gulf between rationalized experiments in biochemistry and the disorderly complexity of the cell has long seemed impassable. Now writing Nature, Sharma et al.² report a technique that allows 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 linked by various RNA binding proteins (RBP), which control almost every aspect of the mRNA life cycle – from the initial processing of newly made RNAs to their eventual destruction.³. Each RBP can bind to hundreds of RNA molecules and, in turn, each RNA can be bound by dozens of different RBPs⁴. In addition, RNA-protein interactions are not static^5,6. Instead, proteins can quickly bind to their target RNAs and dissociate from them just as quickly (Fig. 1), and this dynamic is 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 to fully understand the regulation of gene expression.

Although RNA-protein interactions have been studied for decades, their kinetics in cells have not been characterized. In general, kinetic vision is only available from in vitro studies using purified proteins; cell experiments identified the target RNAs for RBP, but lacked precision to measure the kinetics of interactions⁵. With the advent of high throughput sequencing methods, in vitro approaches can now probe the kinetics of interactions of a protein with tens of thousands of RNA variants⁷. But these experiments are always carried out on purified proteins in the absence of the cellular medium. In recent years, a method called crosslinking and immunoprecipitation⁸ (CLIP) has become a working tool for the characterization of RNA-protein interactions in cells. In CLIP, a protein in complex with an RNA molecule is covalently crosslinked 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 environment of the cell, but it only provides, at best, a snapshot of these interactions.

Sharma and her colleagues are now bridging 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 key point of the authors was that some technical aspects of the previously reported CLIP methods precluded such approaches from being useful for capturing kinetic parameters. The most difficult limitation is that the cross-linking rates have to be rapid 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 picture. This awareness led the authors to use a pulsed femtosecond UV laser, which crosslinks proteins into RNA quickly enough to capture kinetic parameters. They call their method KIN-CLIP (for kinetics 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 expression.⁹. Dazl binds to hundreds of target mRNAs, increasing their stability and the number of proteins produced^ten. However, despite its biological importance, much about Dazl’s binding and function is unknown, making it an ideal candidate for KIN-CLIP experiments.

Sharma and her colleagues first verified that KIN-CLIP identified RNA targets found in previously published datasets produced from the “snapshot” CLIP. They then calculated kinetic parameters, called 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 resides on individual sites for only a few seconds. Dazl also rarely binds, so the binding sites are free of protein most of the time.

The authors also found that several Dazl molecules tend to bind to sites close to each other. Kinetic analysis suggests that this could be due to cooperative binding – a phenomenon in which the binding of a protein to one site increases the likelihood that other proteins will bind to neighboring sites. Finally, the authors incorporated the newly determined kinetic parameters of Dazl into a model predictive of its impact on gene expression, thus providing a biochemical basis for its function and setting the stage for future research.

One of the most exciting aspects of this study is the potential of KIN-CLIP for studying other RBPs, but the method has some limitations. For example, as with all CLIP-based techniques, the ability to crosslink the protein of interest to bound RNAs is a requirement; this can be difficult, as some proteins do not have the necessary side chains correctly oriented for crosslinking. The biggest hurdle, however, for potential KIN-CLIP converters is that specialized equipment is required for crosslinking: pulsed femtosecond lasers might not be readily available 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 could prove to be another barrier to adoption.

Nonetheless, this study introduced the tools of biochemistry into living cells and in doing so could 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 is also shining on the horizon. Indeed, the authors intriguingly note that pulsed femtosecond lasers can cross-link proteins to DNA – perhaps a “DNA KIN-CLIP” is at hand. Sharma and her colleagues not only set a new standard in RNA biology, they may also have unleashed the power of biochemistry over molecular biology more generally.