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GE Healthcare Application Note 16 Biacore systems Unraveling the mechanisms of RNA-binding protein functions using SPR-based kinetic analysis • New insights into how proteins interact with RNA Introduction • Dynamic biomolecular interactions investigated using rate constant analysis The association of specific binding proteins with RNA molecules begins immediately as the nascent RNA chain is produced during transcription. From this point onwards, a series of different proteins are bound to and released from the RNA and are involved in co- and post-transcriptional RNA processing events such as splicing, export to the cytoplasm, regulation of RNA stability and translation [1]. These interactions are highly dynamic and are critical in the regulation of the various steps involved in RNA processing. In addition, these individual RNA-protein interactions occur in the environment of extremely large, multi-component nucleic acid/protein complexes such as the transcriptosome and spliceosome. • Two-step model proposed for U1A binding to RNA Abstract Most RNA molecules are functionally dependent on their associations with a range of different RNA-binding proteins. These interactions are highly dynamic in nature and the balance between binding and dissociation events is likely to play a pivotal role in the function of RNA-binding proteins. Biacore™ systems are ideally suited to the detailed kinetic analysis of biomolecular interactions and have been instrumental in gaining mechanistic insights into the action of key RNA-binding proteins. The interactions of the neuronspecific RNA-binding protein, HuD, and the spliceosomal protein, U1A, with their target RNAs have been studied. The evaluation of individual rate constants provides important new insights into RNA/protein interactions. In addition to greatly facilitating the understanding of how these two important RNA-binding proteins function, this work clearly demonstrates that steady-state affinity studies are not sufficient to describe this type of complex biomolecular interaction. From the above considerations, it may seem obvious that any attempt to understand these complex processes demands a detailed knowledge of the dynamic binding characteristics of individual protein-RNA interactions. Until recently, however, studies on RNA-binding proteins have relied heavily on techniques that examine binding under equilibrium conditions (such as electrophoretic mobility shift and filter binding assays). Although this type of affinity-based data is valuable in many instances, it does not address the key issue of the dynamics of the RNA-protein interactions. This requires a true kinetic analysis that can deliver individual association and dissociation rate constants (ka and kd). Acknowledgement We gratefully acknowledge the valuable cooperation of Dr. Ite Laird-Offringa and the members of her laboratory in preparing this Application Note. This Application Note, based on the work of Dr. Ite LairdOffringa’s group [references 2–5], describes how Biacore systems can be used to provide detailed kinetic data on the interaction of RNA-binding proteins with their specific RNA targets and shows how this information can provide important insights into their functions. The binding of the neuronal protein HuD to RNA is shown to require the activity of three different RNA-binding domains, which provide their own distinct contributions to the overall kinetics of the binding interaction [2]. Detailed kinetic analysis is also used to demonstrate that the binding interaction between the spliceosomal protein U1A and its RNA target occurs by a twostep mechanism [3]. In addition to describing the results from these important studies, practical information on optimizing RNA-protein studies is presented. Methods Detailed descriptions of experimental and data evaluation methods used in the HuD and U1A studies can be found in references [2] and [3]. Some important general issues in the experimental design of RNA-binding protein studies are discussed. For further details, the reader is directed to a more extensive discussion of these issues in reference [4]. Choice of immobilized interaction partner (ligand) In RNA-protein studies the nucleic acid is most often immobilized and oriented onto a streptavidin sensor surface (Sensor Chip SA) due to the ease with which RNA oligonucleotides can be chemically synthesized with a terminal biotin modification. In this case, it can be advantageous to include a few extra nucleotides as a “spacer” to minimize any possible steric hindrance effects that could interfere with the interacting molecules. If longer RNAs are produced by in vitro transcription, these should be gel-purified. An alternative strategy for capturing RNA on the sensor surface is to anneal it to a complementary single-stranded DNA oligonucleotide that is immobilized via a biotin group. This approach is achieved by extending the target RNA with additional nucleotides that are complementary to the immobilized DNA oligonucleotides, using cloning sitederived sequences from the plasmid, for example [2]. These alternative strategies for immobilizing RNA are illustrated in Figure 1. Depending upon the particular application, secondary structure within the target RNA may or may not be desired and these requirements can be addressed by careful oligonucleotide design and choice of experimental conditions. Secondary structure can be avoided by designing RNA 2 05/2007 28-9213-56 AA U AUCCA U U GCACU C C G U AUCCA U U GC ACU CC G G ACUCUAGAGGAUCCCGG C TGAGATCTCCTAGGGCC Figure 1. Alternative strategies for immobilizing RNA on the sensor surface. A. RNA is immobilized directly on the streptavidin moieties on the surface of Sensor Chip SA via a 5’ biotin group. B. A biotinylated, single stranded DNA oligonucleotide is first immobilized on the streptavidin surface. A complementary sequence present on the 3’ end of the RNA molecule is then used to anneal the RNA to the oligonucleotide, leaving the proteinbinding region available for interaction. RNA sequences are shown in red and DNA sequences in black. sequences that lack any complementarity between the target region and other parts of the molecule. If partially double-stranded regions are required, however, these can be made more stable by extending the complementary sequences to increase base pairing. Heating and slow cooling of the RNA prior to application to the sensor surface will also help to promote secondary structure formation. Immobilizing RNA The optimal level of immobilized ligand depends upon the size of the analyte (which dictates SPR signal per bound molecule) and the type of data required. For kinetic experiments, a relatively low Rmax (theoretical saturated response) is generally desirable and in the studies described in this Application Note, RNA immobilization densities of 25–30 RU (response units) and 100–125 RU were used for strongly and weakly binding protein analytes, respectively [2, 3]. Sensor Chip SA is conditioned with three consecutive injections of 1M NaCl/50 mM NaOH (100 µl at 50 µl/min) to remove any unconjugated streptavidin. Biotinylated RNAs are prepared as a 1µM solution in running buffer (10 mM TrisHCl, pH 8, 150 mM NaCl), heated to 80°C for 10 minutes and cooled slowly to room temperature. The RNA solution is then diluted 500-fold in Tris/NaCl running buffer supplemented with 0.005% surfactant P20, 62.5 µg/ml BSA, 125 µg/ml tRNA, 1 mM DTT and 5% glycerol. Immobilization is then carried out using a series of 10–20µl injections at a flow rate of 10µl/min, repeated until the desired immobilization level is achieved. If non-biotinylated RNAs are used, a biotinylated, partially complementary single-stranded DNA molecule of around 20 nucleotides in length is first attached to the sensor surface, to a level of approximately 60 RU. RNA (0.5µM in 1M NaCl) is then injected at a low flow rate (2µl/min) to allow annealing of the RNA to the DNA via their complementary regions. Annealing the nucleic acids on the sensor surface, rather than prior to immobilization also enables real-time SPR monitoring of the hybridization process itself. Regeneration conditions and experimental controls Regeneration requirements for RNA sensor surfaces follow general principles for interaction analysis i.e. to remove bound analyte from the sensor surface between sample injections without affecting ligand activity. When studying protein binding to immobilized RNA, a regeneration injection of 2M NaCl for 1 minute at a flow rate of 20 µl/min generally works well. The choice of reference surface largely depends upon the immobilization strategy. If biotinylated RNA is captured directly on Sensor Chip SA, an uncoated SA surface acts as a suitable reference. If the indirect capturing approach is used, however, a reference flow cell surface should be prepared by immobilizing a comparable amount of biotinylated DNA oligonucleotide as used in the measurement flow cell. These reference surfaces will provide a check for any nonspecific binding events, but the latter should be minimized during assay development steps for any given application. The modified running buffer (as described previously for RNA immobilization) used for the HuD and U1A studies was optimized to minimize non-specific binding and protein aggregation. HuD, like all the members of this protein family, contains three conserved RNA recognition motifs (RRMs). Work using partially purified RNA targets and equilibrium analysis methods had suggested that only two of the three RRMs were required for binding. A quite different picture was seen, however, when SPR studies were carried out using short AU-rich RNA oligonucleotide targets [2]. These RNAs were immobilized on the sensor surface via annealing to partially complementary, biotinylated DNA oligonucleotides. Using HuD variants containing specific deletions of each RRM, the detailed kinetic information obtained using Biacore X shows that in fact, all three RNA-binding regions are important in forming a stable RNA/protein complex (Figure 2). Affinity Association Dissociation Mutations of HuD 1 2 3 1 2 3 1 2 1 2 wt wt wt 2000 x 20 x 100 x 3 13.5 x 2.5 x 35 x 3 3.5 x 4x 14 x Figure 2. Effects of RRM deletions on affinity and kinetic characteristics of HuD/RNA binding. The three RNA recognition motifs of wild type HuD are indicated by the three numbered boxes. Deletion mutants are indicated. Approximate relative changes in affinity and association/dissociation rate constants are indicated in the table (“wt” indicates reference parameters derived from wild type HuD). While the affinity data correlates with previous studies showing that RRM1 is the major RNA binding region, RRM2 plays an intermediate role and that deletion of RRM3 has only a marginal effect on affinity, these steady-state measurements mask major kinetic contributions by the individual RNA-binding domains. In particular, the dissociation rate constants (k ) obtained show that deletion of either RRM2 or RRM3 results in a significant increase in dissociation rate (i.e., a destabilization of the protein-RNA complex). These effects are masked in terms of affinity measurements, however, since they are accompanied by parallel increases in the association rates of these deletion mutants (Figure 2). This is in contrast to the deletion of RRM1, which produces a simultaneous decrease in association rate and increase in dissociation rate, resulting in the dramatic reduction in overall affinity. d Results Unmasking the roles of multiple RNA recognition motifs in the binding of HuD to AU-rich RNA HuD is a member of the Hu family of highly conserved RNA-binding proteins, all of which bind to AU-rich sequences in mRNAs. Three of the four known Hu proteins are expressed specifically in neurons and are believed to be involved in neuron-specific post-transcriptional gene regulation. These functions may involve the effects of Hu proteins on mRNA stability. The AU-rich sequences to which these proteins bind are frequently found in the 3´ untranslated regions of labile mRNAs, such as those from growth factor genes. These results indicate that although RRM2 and particularly, RRM3 do not have a major effect on overall affinity, they have very significant effects on kinetic binding behavior. This is likely to be a vital component of RNA-binding protein function in vivo, a view that is supported by functional studies showing that Hu proteins lacking RRM 2 or 3 lose their biological activity. 05/2007 28-9213-56 AA 3 Kinetic analysis reveals a two-step mechanism for the binding of U1A to its target RNA U1A is a spliceosomal protein that binds specifically to a stem-loop structure within the U1 small nuclear ribonucleoprotein RNA (U1hpII). Although U1A contains two RRM-type domains, previous studies have determined that only the N-terminal RRM is involved in binding to U1hpII. Despite a considerable body of structural and biochemical data, however, a kinetic (and hence mechanistic) description of this system has remained elusive. Using the co-crystal structure of U1A/U1hpII to design experimental mutants of both U1A and its target RNA, Katsamba et al [3] have used SPR-based kinetic analyses to propose a novel, two-stage binding mechanism. Using Biacore 2000 and Biacore 3000, biotinylated wild-type and point-mutated U1hpII RNA targets were immobilized on the sensor surface and analyzed for binding to a series of U1A variants containing targeted amino acid substitutions. Wild type U1A and U1hpII were shown to bind with pM affinity (in agreement with previous studies) and kinetic determinations revealed that this derived from a combination of both a rapid association (ka = 1.1 × 107 M-1s-1) and slow dissociation (kd = 3.6 × 10-4 s-1). Since the rapid association rate suggested the involvement of electrostatic interactions in the binding of U1A to its negatively charged RNA target, the role of basic amino acids in the vicinity of the RNA binding pocket was examined. The co-crystal structure of U1A/ U1hpII was used to select positively charged residues that were appropriately located, but which were not implicated in hydrogen bonding interactions. Three conserved lysine residues (20, 22 & 50) were consequently targeted to make alanine substitutions, generating Lys20,22Ala (double mutant) and Lys50Ala (single mutant) U1A variants. Kinetic analyses showed that both of these mutants exhibited a significant loss of affinity compared to wild type U1A and that in both cases, this was predominantly due to a reduction in association rate (Table 1). This data also showed a moderate increase in dissociation rate for Lys20,22Ala U1A, whereas the effect of the Lys50 mutation on dissociation rate was minimal. This demonstrates that the three lysine residues play an important role in bringing the RNA and protein together, although Lys20/Lys22 also have a moderate additional role in maintaining complex stability. 4 05/2007 28-9213-56 AA Table 1. Kinetic and affinity effects of lysine-to-alanine substitutions in U1A on its binding to U1AhpII. The equilibrium dissociation and association/ dissociation rate constants are given for wild type U1A, whereas the relative changes in affinity, association and dissociation compared to wild type U1A are shown for the mutants. protein affinity association rate dissociation rate wt U1A KD = 32 pM ka = 1.1×107 M -1S -1 kd = 3.6×10-4S -1 Lys20,22Ala 39 × 9.8 × 3.9 × Lys50Ala 16 × 10 × 1.5 × Table 2. Kinetic and affinity effects of increased salt concentration on wild type and mutant U1As. The changes in affinity and kinetics shown represent the relative values at 500 mM NaCl compared to those obtained for the same protein in standard binding buffer (150 mM NaCl). protein affinity (500 mM NaCl) association rate (500 mM NaCl) dissociation rate (500 mM NaCl) wt U1A 132 × 59 × 2.2 × Lys20,22Ala 108 × 14 × 7.6 × Lys50Ala 136 × 19 × 7.3 × This role for positively charged lysine residues in association of U1A with the RNA target implies that electrostatic interactions are involved; the ionic microenvironment would therefore be expected to have a significant effect on binding. As shown in Table 2, increasing the NaCl concentration of the binding buffer from 150 to 500mM reduced the affinity of the U1A/U1hpII interaction by more than 100fold. Measurements of the individual rate constants further showed that this loss of affinity was over-whelmingly due to a decrease in association rate. Although as expected, increasing salt concentration also reduced the association rates for the lysine mutant U1A variants (each of which contained intact basic residues), this was significantly less marked compared to the wild-type protein. Taken together, the mutational and salt concentration kinetic data confirm that three lysine residues in the vicinity of the RNA-binding pocket of the U1A/U1hpII complex play a major role in its initial association and that this involves electrostatic interactions. Table 3. Kinetic and affinity effects of mutations in the protein/RNA interface of the U1A/U1hpII complex. Phe56Ala and U1hpIIG4C are the U1A and RNA mutations described in the text. The affinity and kinetic values shown for each mutant were obtained from SPR measurements of their binding to the appropriate wild type interaction partner and are relative to those of the wild type U1A/U1hpII interaction. mutation affinity association rate dissociation rate Phe56Ala 6600 × 4.7 × 1400 × U1hpllG4C 8700 × 3.5 × 2500 × Kinetic analyses of both protein and RNA mutations designed to study U1A/U1hpII complex stability were also performed. The co-crystal structure identified a particular base (A6) in the RNA loop that stacks with U1A residue Phe56. This requires a rearrangement from the free protein, however, suggesting a requirement for induced fit of RNA and protein. A Phe56Ala mutant of U1A was consequently generated and this exhibited a 6600-fold decrease in affinity compared to wild type (Table 3). When analyzed at the level of the individual rate constants, however, this dramatic change in affinity was found to derive almost exclusively from an increase in dissociation rate (1400-fold, compared to a decrease of less than 5-fold in association rate). The mutation of nucleotide G4 from the RNA loop of U1hpII (which forms hydrogen bonds in the RNA/protein interface) to a cytosine showed a very similar effect on the affinity and kinetics of binding to wild-type U1A (2500-fold increase in dissociation rate and 3.5-fold decrease in association rate). These data confirm an important role for aromatic stacking and hydrogen-bonding in complex stability, which involves defined amino acid and nucleotide residues in the RNA/protein interface. On the basis of structural comparisons between free U1A and the protein/ RNA complex, the formation of these close-range interactions is thought to occur through an induced fit. The kinetic analysis described above shows for the first time that binding of U1A to U1hpII is a two-step interaction. Moreover, the high-resolution data demonstrate that specific residues within the protein and RNA take part in the initial association and subsequent induced-fit stabilizing phases of the interaction. This led the authors to propose a “lure and lock” mechanism for binding, with two distinct steps that are dominated by electrostatic attraction and close-range aromatic stacking/hydrogen-bonding forces, respectively (Figure 3). 1. “Lure” 2. “Lock” Figure 3. A two step “lure and lock” model for the binding of U1A to U1hpII RNA. During the first step, rapid association occurs via electrostatic interactions between the negatively charged phosphate backbone of the RNA and positively charged amino acid residues around the RNA-binding tract of the protein. Lysine residues demonstrated to play a role in this step are indicated (K20, 22 and K50). In the second step of the model, close-range hydrogen bonding and aromatic stacking interactions, which occur during the induced fit of RNA and protein, lock the binding partners into a stable complex. U1hpII nucleotide G4 and U1A residue Phe56 were shown to be important for this step. Conclusions These studies into the binding mechanisms of the RNAbinding proteins, HuD and U1A, demonstrate the importance of detailed kinetic analyses when investigating dynamic biomolecular interactions. In the case of HuD, contributions made by individual RRMs that had remained “hidden” during previous equilibrium analysis-based studies were shown to make important kinetic contributions to the overall binding function. Many RNA-binding proteins contain multiple RRM regions and the results from the HuD study may therefore represent a general model for how investigation into this type of protein should be approached. The analysis of U1A binding to its target RNA revealed a previously unknown two-step mechanism of binding. This data was also able to identify key amino acid and nucleotide residues involved in the individual interaction stages and provide good evidence for the types of forces involved. This model may also prove to have a much wider relevance, since many other RNA-binding proteins are known to contain basic amino acids distributed around their RNA binding sites. In both cases described in this Application Note, crucial new information regarding the mechanism of the protein-RNA interaction was obtained. These studies relied heavily on high-quality kinetic data and, the authors have recognized the suitability of Biacore instruments for future developments in this field [4]. In addition to furthering our understanding of the many vital biological interactions between proteins and RNAs, kinetic approaches may also be invaluable in designing small molecule inhibitors with considerable therapeutic potential. 05/2007 28-9213-56 AA 5 References 1. Burd, G.C. and Dreyfuss, G. Conserved structures and diversity of functions of RNA-binding proteins. Science 265: 615–21 (1994) 2. Park, S., Myszka, D.G., Yu, M., Littler, S.J. and Laird-Offringa, I.A. HuD RNA recognition motifs play distinctive roles in the formation of a stable complex with AU-rich RNA. Mol Cell Biol 20: 4765–72 (2000) 3. Katsamba, P.S., Myszka, D.G. and Laird-Offringa, I.A. Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA. Biol Chem 276: 21476–81 (2001) 4. Katsamba, P.S., Park, S. and Laird-Offringa, I.A. Kinetic studies of RNAprotein interactions using surface plasmon resonance. Methods 26: 95–104 (2002) 5. Katsamba, P.S., Bayramyan, M., Haworth, I.S., Myszka, D.G. and LairdOffringa, I.A. Complex role of the β2–β3 loop in the interaction of U1A with U1 hairpin II RNA. J Biol Chem 277: 33267–74 (2002) GE Healthcare Biacore AB Rapsgatan 7 754 50 Uppsala Sweden www.biacore.com imagination at work GE, imagination at work and GE monogram are trademarks of General Electric Company. Biacore is a trademark of Biacore AB, a GE Healthcare company. All third party trademarks are the property of their respective owners. © 2004 - 2007 General Electric Company – All rights reserved. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. BR-9002-97 (05/2007) 28-9213-56 AA First published 09/2002