A New Laser-based Approach to Measure RNA-protein Interactions within Live Cells

Gene expression in eukaryotic cells is a complex process that involves a myriad of molecules which are very different in origin, structure, expression kinetics and function. It is a process tightly regulated by the presence of methylation and acetylation marks at given genomic regions that alters chromatin relaxation; by contacts between distal sequences of regulatory DNA regions; by transcription factors, which are proteins that binds to the DNA within regulatory regions and recruits the transcription machinery; and even by RNA regulatory molecules that binds to the DNA and enhance or repress gene transcription. Nevertheless, gene expression is not only controlled at the transcriptional level, but also after transcription in a process called posttranscriptional regulation. After messenger RNA (mRNA) synthesis, several RNA-binding proteins (RBP) can bind to certain mRNAs, and their dynamic of binding and dissociation are critical for the regulation of gene expression [1].  

An example of posttranscriptional regulation, one of the steps that controls mRNA expression. Kojima S, et al. Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expression. Genes Dev. 2012 Dec 15;26(24):2724-36. doi: 10.1101/gad.208306.112.

Classically, the only way to assess the RBP binding and dissociation kinetics was through in vitro experiments, and just steady-state patterns of RNA-protein interactions have been determined in cells so far [2]. Furthermore, the inaccessibility of these kinetics in cells limits the establishment of quantitative connections between RBP-RNA interactions and cellular RBP function. Now, approaches relying in the nanotechnology field of photonics have pave the way for the study of complex molecule interactions. Recently published in Nature [3], Sharma D, et al. developed a time-resolving crosslinking approach called kinetic crosslinking and immunoprecipitation (KIN-CLIP), to measure cellular binding and dissociation kinetics of RNA-protein interactions at individual binding sites on a transcriptome-wide scale. The KIN-CLIP approach uses a femtosecond (fs) ultraviolet (UV) laser that efficiently increases the RNA-protein crosslinking without altering their crosslinking patterns. 

An overview of the KIN-CLIP process and its in vitro validation. A represents the kinetic scheme for RNA-protein binding and crosslinking, and B the reaction scheme. C represents the schematic representation of the KIN-CLIP process and D the comparison between the values calculated with previous methods (such as anisotropy) and the ones obtained with the KIN-CLIP method. Sharma D, et al. The kinetic landscape of an RNA-binding protein in cells. Nature. 2021 Feb 10. doi: 10.1038/s41586-021-03222-x. 

Calculation of rate constants requires a sufficient number of experimental constraints that can be established by measuring crosslinking time courses at different protein concentrations and different crosslinking efficiencies while ensuring that crosslinking rates constant are equal or larger than dissociation and apparent association rate constants. For the validation of the KIN CLIP tool in vitro, authors first determine the binding, dissociation and crosslinking rate constants of the RNA-recognition motif (RRM) of the RBP RBFOX (RFOX(RRM)) at different laser powers and protein concentrations. They found that the apparent affinity of RBFOX(RRM) RNA, calculated from association and dissociation rate constants, was similar to the affinity measured by other in vitro methods [4]. Same in vitro analysis with another two RBPs indicated that binding and dissociation rate constants for RNA-protein interactions can be determined by time-resolved crosslinking with a fs laser. 

Next, they validated the KIN-CLIP tool by measuring the RBP kinetics within live cells. Authors used a RNP called Deleted in Azoospermia Like (DAZL) that is essential for eukaryotic gametogenesis [5] and contains one RRM, binds predominantly to the 3’-Untranslated Region (3’-UTR) of mRNAs and regulates mRNA stability and translation [6]. The protein was expressed in mouse GC-1 cells under the control of a doxycycline-inducible promoter, so changes in doxycycline concentration allowed measurements at different concentrations of DAZL. DAZL crosslinking sites measured with KIN-CLIP were identical to those observed with previous techniques, maintaining the integrity of the binding site. For most binding sites the association rate constants at a lower DAZL concentration were lower than those with higher concentration, indicating that only a small fraction of mRNA binding sites are saturated with DAZL at lower protein concentrations. The kinetic data reveal highly dynamic DAZL-RNA interactions, with most DAZL-binding events being rare and transient.

DAZL-binding sites identified by KIN-CLIP (fs laser) and conventional techniques such as UV crosslinking (iCLIP) on all RNAs and 3’ UTRs. Sharma D, et al. The kinetic landscape of an RNA-binding protein in cells. Nature. 2021 Feb 10. doi: 10.1038/s41586-021-03222-x.

Researchers also wanted to understand how DAZL regulates mRNA function by examining the patterns of the kinetic parameters for all DAZL-binding sites on bound mRNAs. They found that the majority of DAZL-binding sites are located in the 3’-UTR of target mRNAs, frequently proximal to the polyadenylation site (PAS), and that most DAZL-bound mRNAs contained multiple DAZL-binding sites with an inter-site distance markedly smaller than the expected by a stochastic event, even when distant from the PAS. They proposed that there is a clustering of multiple DAZL-binding sites on most 3’-UTRs, and that the number of binding sites within a 3’ UTR cluster increased with proximity to the PAS. Binding site context, which includes the RNA structure and proximal binding of other proteins, may influence the accessibility of binding sites within a cluster, being another factor that must be taken into account for clustering studies. 

In sum, the KIN-CLIP technique reveals a highly dynamic in the RNA binding of DAZL, which resides at individual binding sites for time periods even shorter than seconds, remaining cognate sites DAZL-free most of the time. It has been described that cellular concentrations of DAZL are sub saturating relative to its RNA targets, and its highly dynamic binding allows rapid changes in the patterns of RNA binding that could be critical for its function. Furthermore, many RBPs and some regulatory RBPs also bind their cognate RNA sites transiently and infrequently, and occasionally none might be bound to a given mRNA at the same time. Cellular kinetic data will be also important for decoding the complex link between patterns of DAZL RNA binding and DAZL function.    

In addition to RNA-protein interactions, KIN-CLIP can be used for the biochemical characterization and quantification of other RNA-protein interactions in cells, or even to assess the interaction between other biomolecules such as DNA-proteins, proteins-proteins, proteins carbohydrates, and so on. This technology is an excellent example of how nanotechnologies (in this case, photonics) can be merged to biology to depict an outstanding landscape of the complex regulation of gene expression.

References:

1. Licatalosi, D.D., X. Ye, and E. Jankowsky, Approaches for measuring the dynamics of RNA-protein interactions. Wiley Interdiscip Rev RNA, 2020. 11(1): p. e1565.

2. Corley, M., M.C. Burns, and G.W. Yeo, How RNA-Binding Proteins Interact with RNA: Molecules and Mechanisms. Mol Cell, 2020. 78(1): p. 9-29.

3. Sharma, D., et al., The kinetic landscape of an RNA-binding protein in cells. Nature, 2021.

4. Auweter, S.D., et al., Molecular basis of RNA recognition by the human alternative splicing factor Fox-1. EMBO J, 2006. 25(1): p. 163-73.

5. Fu, X.F., et al., DAZ Family Proteins, Key Players for Germ Cell Development. Int J Biol Sci, 2015. 11(10): p. 1226-35.

6. Yang, C.R., et al., The RNA-binding protein DAZL functions as repressor and activator of mRNA translation during oocyte maturation. Nat Commun, 2020. 11(1): p. 1399.