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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
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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.
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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.
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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.
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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)
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BR-9002-97 (05/2007)
28-9213-56 AA First published 09/2002