Development of FRET based assays
The distance dependent nature of fluorescence resonance energy transfer (FRET) is ideally suited to monitor assembly of the chaperone complex and formation of the U3-18S and U3-ETS duplexes. In our steady-state FRET (ssFRET, i.e. with continuous illumination and observation) assays one molecule is labeled with the donor fluorescein (Fl) and its potential partner with the acceptor tetramethylrhodamine (Rh). When partner macromolecules interact they produce a ssFRET value above background if the fluorophore pairs are sufficiently close (between ~15 and 80 Å). To confirm that the fluorescent labels do not interfere with binding activity, we determined that the RNA-protein Kd
values using fluorescently labeled molecules (data not shown) were within a factor of two of those measured previously with radiolabeled RNA and unlabeled protein16
. To determine the duplex association (kon
) and dissociation (koff
) rate constants we monitored the signal change associated with the donor emission because it is larger than that of the acceptor emission. This phenomenon is due in part to more FRET-independent crosstalk from the donor to the acceptor than vice versa because of the asymmetry of their emission peaks. Three lines of evidence provide confidence that the Fl signal monitors duplex formation in accord with FRET. First and foremost, addition of acceptor containing RNA molecules results in a decrease in the Fl peak emission with concomitant increase in Rh peak emission for each case studied, whereas addition of an unlabeled partner to the Fl-labeled U3 MINI does not quench donor emission (Supplementary Fig. S1
). Second, duplex kon
values were within a factor of two of those determined with fluorophores attached to different sites on the RNA substrates (data not shown). Third, the Kd
calculated by dividing U3-18S duplex koff
by its corresponding kon
is within a factor of two of the mean Kd
value measured by electrophoretic mobility shift assays (; Supplementary Fig. S2
U3-18S duplex kinetic and thermodynamic parameters
To distinguish the U3-ETS duplex parameters from those of the U3-18S duplex, the former are designated henceforth kon (ETS), koff (ETS) and Kd (ETS) and the latter kon (18S), koff (18S) and Kd (18S).
Chaperone complex includes U3 MINI, Imp3p and Imp4p
Our previous findings16
showed that Imp3p and Imp4p share the same minimal RNA binding site, U3 MINI. To test whether these proteins assemble with U3 MINI into a ternary chaperone complex, we used ssFRET assays in which Fl-labeled Imp3p (Fl-Imp3p) contained the donor, Rh-labeled Imp4p (Rh-Imp4p) contained the acceptor, and U3 MINI was unlabeled (). Addition of Rh-Imp4p to a pre-formed binary complex of Fl-Imp3p and U3 MINI resulted in a FRET efficiency (EFRET
, calculated as described in Materials and Methods) value of 0.26 ± 0.01, significantly above background (0.03), consistent with assembly (, open bar). In contrast, only background EFRET
values were observed when Fl-Imp3p was added to either Rh-Imp4p (, hashed bar) or a pre-formed binary complex of Rh-Imp4p and U3 MINI (, black bar). Addition of unlabeled Imp4p to a preformed binary complex of Fl-Imp3p and U3 MINI showed no signal change and thus confirmed that the observed FRET signal results from the proximity of the Rh and Fl labels and not protein binding (). These findings support the notion that an RNA-dependent chaperone complex assembles from U3 MINI, Imp3p and Imp4p and lead to the hypothesis that Imp3p binds to U3 MINI before Imp4p.
Fig. 2 Assembly of the chaperone complex between Imp3p, Imp4p and U3 MINI is RNA-dependent and may be ordered. (a) EFRET values are shown for three conditions: addition of Fl-Imp3p to a preincubated mixture of Rh-Imp4p and U3 MINI (filled bar); a mixture of (more ...)
To verify assembly of the chaperone complex with full length U3 snoRNA, metal affinity chromatography was used to capture N-terminal His6–tagged Imp3p (His6-Imp3p) in the presence of untagged Imp4p and full-length U3 snoRNA (). Denaturing PAGE analysis of the loaded mixtures and eluted fractions after washes that include high salt (1 M NaCl) shows that His6-Imp3p and Imp4p associate with each other in the presence of U3 snoRNA (, lane 2). To confirm that retention on the column arises from interaction with the tagged protein, we showed that neither the unlabeled Imp4p nor U3 snoRNA remain bound after the washes (, lane 4).
Our in vitro
assembly findings are consistent with previous immunoprecipitation studies using S. cerevisiae
cell extracts, which showed that prior binding of Imp3p is needed to incorporate Imp4p into the SSU processome22
. Such correlation between our in vitro
studies and immunoprecipitation assays of others helps validate our in vitro
system as biologically relevant.
Defining and examining a duplex formation framework
To ascertain the limits to duplex yield and hybridization rate constants we investigated how addition of protein affects the duplex stability (Kd) and duplex kinetics (kon and koff). Evaluation of these effects will also discriminate between the six mechanistic models that are envisioned to overcome these limitations (). Given that Kd = koff/kon, we determined any two of these values, which are sufficient to calculate the third value for a single step model of reversible duplex formation as illustrated by the U3-ETS duplex (). In contrast, formation of the U3-18S duplex is most readily modeled with two discernable steps: unfolding of the box A/A’ stem structure and subsequent hybridization (). For the first (unfolding) step, we estimated the Keq equilibrium constant between U3 MINI and its unfolded form, designated U3 MINI*, and how protein binding affects this constant. For the second (hybridization) step in U3-18S duplex formation, appropriate conditions were used to determine kon (18S) and koff (18S) directly rather than Kd (18S). The binding affinity is expected to include contributions from both the unfolding and hybridization steps resulting in an apparent Kd (18S) ().
Fig. 3 Free energy reaction profiles illustrate the six possible mechanisms used by proteins to mediate duplex formation by changing the energy levels of the substrate, transition state, product or some combination thereof. Evaluation of how the magnitude of (more ...)
Stability of the box A/A’ stem structure hinders U3-18S duplex formation
Chaperone activity can mediate U3-18S duplex formation by affecting the unfolding step, the hybridization step or both. We begin by investigating the unfolding step (). To place an upper estimate on the free energy of unfolding the U3-stem structure we obtained reversible UV melting data for U3 MINI from S. cerevisiae (). The melting temperature of 54 °C corresponds to a free energy of 4 kcal/mol with a Keq of 10−3 at 20 °C. As this stem structure is conserved among eukaryotes, it is expected to remain folded even at the growth temperature of vertebrates (37 – 42 °C) with only trace quantities (~0.1%) of U3 MINI*, the unfolded form of U3 MINI. To ensure rapid formation of the U3-18S duplex, helix destabilization activity is thus anticipated.
Fig. 4 The stable box A/A’ stem structure of U3 MINI is unfolded to U3 MINI* by addition of protein, primarily Imp3p. (a) OD260 values for unlabeled U3 MINI melted in the forward (circles) and refolded in the reverse direction (squares). A smoothed derivative (more ...)
Assembly of the chaperone complex opens up the U3-stem structure
To test whether assembly of the chaperone complex opens up the U3-stem structure and thereby changes Keq
, we used time-resolved FRET (trFRET). Unlike ssFRET, trFRET23,24
compares the nanosecond-scale donor fluorescence decay in the presence and absence of the acceptor to determine with high precision the distribution of distances separating the fluorophore pair. We measured trFRET of a doubly labeled U3 MINI with Fl at the 5’ end and Rh on the opposite side of the box A/A’ stem (Fl-U3 MINI-Rh, ) in the presence and absence of proteins and 18S (decay curves shown in Supplementary Fig. S3
). Determination of the Fl-Rh distance distributions in U3 MINI alone showed that 93% of the RNA molecules yield a short (~19 Å) mean Fl-Rh distance (, grey line), as expected from a donor and acceptor on opposite sides of an A-form helical RNA (). The remaining 7% of RNA molecules reside in a conformation of larger and more broadly distributed Fl-Rh distances (, grey dashed line), consistent with the presence of a small fraction of U3 MINI dimer (, inset).
Upon addition of Imp3p to Fl-U3 MINI-Rh (, dashed black line), the mean Fl-Rh distance increases by 13 Å from 19 Å to 32 Å with a concomitant sharpening of the distance distribution (, compare the solid grey and dashed black lines). Subsequent addition of Imp4p and 18S results in only minor changes (, compare the black line with the dashed and dotted black lines) supporting the view that Imp3p is primarily responsible for unfolding U3 MINI to U3 MINI*.
The 13 Å increase readily accommodates an open box A/A’ stem structure but not a fully extended U3 MINI* that could separate the Fl-Rh pair by as much as 100 Å, and thus abolish FRET. To account for the lack of change in distance distributions upon addition of Imp4p and 18S, our data are most consistent with a model in which the 3’ segment of U3 MINI loops back as shown in , as a result of Imp3p binding. This arrangement remains unchanged upon addition of Imp4p and 18S. Our trFRET data suggest that assembly of the chaperone complex mediates the first unfolding step by opening up the box A/A’ stem structure.
The chaperone complex accelerates U3-18S duplex formation by unfolding U3 MINI to U3 MINI* not by stimulating kon (18S)
To test whether the chaperone complex affects the second U3-18S hybridization step, we investigated how the kon (18S) and koff (18S) change upon addition of Imp3p and Imp4p (; ). We determined the kon (18S) by monitoring the time-dependent donor quenching of 5’-Fl-labeled U3 MINI (Fl-U3 MINI) upon addition of equimolar amount of 3’-Rh labeled 18S (18S-Rh) in the presence of saturating amounts of protein. This stoichiometry was used to ensure a 1:1 donor-to-acceptor ratio for a maximum ssFRET signal change. Duplex formation with a kon (18S) of (7 ± 1) × 105 M−1s−1 was observed only after assembly of the chaperone complex (, black squares, and c). To ensure that this rate directly monitors the bimolecular hybridization step we verified that this kon (18S) was the same, within error, as that determined using more conventional pseudo first-order conditions (excess 18S-Rh, ).
Fig. 5 Hybridization kinetics of the U3-18S duplex reveal that kon (18S) does not limit duplex formation. (a) The substrates used for kinetic measurements were the 5’-Fl labeled U3 MINI (Fl-U3 MINI) and the 3’-Rh labeled 18S (18S-Rh). The dashed (more ...)
In sharp contrast to rapid U3-18S hybridization in the presence of protein, duplex formation was not detectable in the absence of protein even when up to 1 µM concentrations of 18S-Rh were used (, compare the traces with open and grey circles). Likewise, no shift was detected with electrophoretic mobility shift assays using up to 200 µM U3 MINI with trace amounts of 32P-18S (data not shown).
To place an upper estimate on kon
(18S) after the box A/A’ stem has opened up, we used a fragment of U3 MINI, designated MINI-17, which retains only the 17 nucleotides involved in the U3-18S duplex, including the mismatches (the dashed box in shows the MINI-17-18S duplex). The deleted flanking nucleotides of U3 MINI remove the 3’ half of the box A/A’ stem structure and thus eliminate the need to unfold this structure prior to U3-18S hybridization (). In the absence of protein, the kon
(18S) for MINI-17 hybridizing with 18S is (7 ± 1) × 105
(Supplementary Fig. S4
; ), identical to the kon
(18S) observed for U3 MINI in the presence of protein (). The equivalence of these rate constants supports the view that protein binding has removed the barrier to U3-18S duplex formation by unfolding the box A/A’ stem structure to expose the base-pairing site.
To determine the duplex koff (18S), we chased the pre-formed fluorescently labeled U3-18S duplex with at least 100-fold excess of unlabeled 18S. The time-dependent exponential increase in Fl emission was used to determine that koff (18S) is (2 ± 1) × 10−3 s−1 in the presence of protein (). In the absence of protein, koff (18S) was not measured because formation of this duplex was not observed. To estimate koff (18S) in the absence of protein, we therefore determined koff (18S) for the MINI-17 – 18S duplex; the observed rate constant of (1.0 ± 0.1) × 10−4 s−1 is 20-fold slower than the U3-18S duplex dissociation rate constant in the presence of protein ().
Fig. 6 Assembly of the chaperone complex increases koff (18S). (a) Representative trace is shown for a chase initiated with 500-fold excess (5 µM) of unlabeled 18S to a preformed duplex between 10 nM Fl-U3 MINI and 10 nM 18S-Rh in the presence of saturating (more ...)
Before comparing these different substrates (U3 MINI and MINI-17), it is useful to consider the step that limits formation of other short duplexes. Classic kinetic studies have shown that hybridization of complementary nucleic acid strands proceeds via two steps: nucleation and elongation25
. Once diffusion juxtaposes bases from two complementary strands, formation of 3 to 4 contiguous base pairs is sufficiently long-lived to nucleate the process. Elongation completes hybridization of the remaining base pairs that flank the nucleation site. Nucleation, not elongation, limits hybridization of two complementary and unstructured RNA strands to form duplexes from 8 to ~20 base pairs in length26
. Consequently, they share the same duplex kon
, independent of their sequence27,28
. Equivalent kon
values are observed for formation of two short duplexes studied herein: the U3-18S duplex in the presence of Imp3p and Imp4 and the MINI-17–18S duplex in the absence of protein. Given the common kon
value it is reasonable to expect that formation of these duplexes is also limited by nucleation.
As a result, comparing how kon (18S) and koff (18S) differ for the U3-18S duplex in the presence of Imp3p and Imp4p and for the MINI-17–18S duplex in the absence of protein offers insight into the mechanism of the hybridization step. Addition of Imp3p and Imp4p does not change the duplex kon (18S) whereas koff (18S) increases by 20-fold, corresponding to a 1.7 kcal/mol destabilization of the U3-18S duplex product (, compare superimposed dotted green (MINI-17-18S duplex) and black (U3-18S duplex) lines). Of the six possible mechanisms, only product destabilization increases koff (18S) and Kd (18S) without changing kon (18S) (; ). The kinetic findings provide evidence that Imp3p and Imp4p do not affect the forward hybridization barrier because kon (18S) remains unchanged but they do destabilize the product duplex after it is formed.
Fig. 7 Reaction profiles illustrating a model for how protein binding accelerates U3-18S duplex formation by unfolding U3 MINI to U3 MINI* (the first step) rather than stimulating hybridization (the second step). In the absence of protein (dashed grey line), (more ...)
The findings from trFRET, UV melting and kinetic studies suggest that protein binding accelerates formation of the U3-18S duplex by unfolding U3 MINI to U3 MINI* (the first step) instead of stimulating annealing activity (the second step). In the absence of protein, two factors limit the amount of U3 MINI* and the subsequent U3-18S duplex (, grey dashed line). First, the 4 kcal/mol stability of the box A/A’ stem structure limits the percentage of U3 MINI* to ~0.1 % (Keq
; ). Second, entropy favors U3 MINI* refolding to U3 MINI rather than bimolecular hybridization. As a result, the protein free reaction is unfavorable. In contrast, trFRET data show that U3 MINI* is the only species detected upon addition of Imp3p (Keq
1), with negligible differences observed upon subsequent addition of Imp4p and 18S (). By increasing Keq
1 assembly of the chaperone complex unfolds U3 MINI into a stable U3 MINI* to accommodate annealing with 18S and ensures that the reaction proceeds energetically downhill from U3 MINI to U3 MINI* to the U3-18S duplex, in contrast to the protein free reaction (, compare grey dashed (no protein) and black lines (protein added)).
Chaperone increases yield of the other hybrid: the U3-ETS duplex
Unlike the kinetic barrier that prevents detectable U3-18S duplex formation in the protein free reaction (), the yield of the other hybrid, the U3-ETS duplex, is limited by thermodynamic instability (). Our previous qualitative findings showed that Imp3p and Imp4p increase the yield of the U3-ETS duplex16
. To quantify the magnitude of this increase we determined the Kd
(ETS) and kon
(ETS) values by using electrophoretic mobility shift assays16
and ssFRET assays, respectively, in the presence and absence of Imp3p and Imp4p (Materials and Methods). Assembly of the chaperone complex decreases the Kd
(ETS) by 100-fold from (7 ± 2) × 10−7
M to (7 ± 3) × 10−9
M (; ), which corresponds to an increase of 2.7 kcal/mol (20 °C) in duplex stability. We determined kon
(ETS) by monitoring the time-dependent donor quenching of the 3’-Fl-labeled U3 MINI (U3 MINI-Fl) upon addition of an equimolar amount of 5’-Rh labeled ETS (Rh-ETS) in the presence and absence of saturating amounts of protein (; ). In contrast to changes in duplex affinity, kon
(ETS) is the same in the presence ((5 ± 1) × 105
) and absence of protein ((6 ± 2) × 105
), within experimental error (P > 0.05) (a representative trace of the no protein reaction is shown in ). The equivalence of these kon
(ETS) values to the intrinsic rate constant for formation of short duplexes27,28
supports the view that hybridization is unhindered even in the presence of Imp3p and Imp4p.
Fig. 8 Chaperone complex stabilizes the U3-ETS duplex without affecting the association rate constant (kon (ETS)). Representative binding data from electrophoretic mobility shift assays for 32P-ETS-U3 MINI duplex in the absence (a) and presence of the chaperone (more ...)
U3-ETS duplex kinetic and thermodynamic parameters
Upon assembly of the chaperone complex, the Kd (ETS) decreases by 100-fold and kon (ETS) remains unchanged, favoring a product stabilization mechanism over the competing alternatives (). A change in Kd rules out transition state stabilization, whereas no change to kon rules out substrate stabilization and destabilization as well as a combined mechanism. By multiplying Kd (ETS) and kon (ETS), koff (ETS) is predicted to increase, which rules out product destabilization. Product stabilization is the only model in which protein decreases Kd without changing kon. The absence of a change to kon (ETS) upon addition of protein reflects an unchanged hybridization barrier (). After hybridization the protein stabilizes this duplex by 2.7 kcal/mol to ensure high yield.
Fig. 9 Reaction profile illustrating a product stabilization model for the U3-ETS duplex mediated by Imp3p and Imp4p in the chaperone complex. The reaction proceeds from U3 MINI (on the left) to the U3-ETS duplex (on the right) with the same barrier height because (more ...)
Our findings support a model in which the product U3-ETS duplex is stabilized by docking into a binding pocket created by Imp3p and Imp4p (). Duplex docking is expected to occur only after the duplex forms because the kon
(ETS) is unaffected by the presence of protein () and the U3 nucleotides involved in hybridization are accessible to ribonuclease digestion16
. A concave binding pocket is an attractive possibility because it most readily accommodates the cylindrical shape of the A-form duplex product.
Chaperone sufficiently enhances formation and yield of both duplexes
By extrapolating our findings using minimal substrates in vitro to the corresponding reactions occurring with full-length pre-rRNA and U3 snoRNA we can estimate whether the chaperone complex satisfies the in vivo demands for rapid formation and high yield of the U3-pre-rRNA duplexes. Addition of the extra pre-rRNA sequences and the numerous trans acting factors found in vivo will undoubtedly affect these results. However, given the many potential complications arising from misfolding of larger RNA substrates, it is important to first determine how RNA chaperones alter duplex formation rates and the yields of minimal RNA substrates.
To calculate duplex rates and yields that simulate in vivo
conditions, the nucleolar concentrations of U3 snoRNA and the pre-rRNA are required. Even though these values are unknown, estimates are possible. High-resolution mapping of rDNA and U3 snoRNA territories in the nucleolus of S. cerevisiae
using optical microscopy indicates volumes of 0.5 × 10−15
L and 1.5 × 10−15
. Given that about 4,000 copies of pre-rRNA30
and between 400 and 1,000 copies of U3 snoRNA31
are expected for rapidly growing cells, the concentration of the U3 snoRNA is between 0.4 and 1 µM and that of the pre-rRNA is ~13 µM. Undoubtedly, the concentration will not be homogeneous throughout the nucleolus; hence our calculations use a broad concentration range from 0.01 to 10 µM. It is also possible to approximate the yield of the U3-pre-rRNA duplexes in vivo
. About 1 in 10 pre-rRNA transcripts are cleaved at A3 before A2 (and A0 and A1; ) resulting in a 23S intermediate rather than the standard 20S precursor (personal communication K. Karbstein, University of Michigan). Because U3-pre-rRNA hybridization is a prerequisite for the A0–A2 cleavages, it is reasonable to assume that these duplexes have not yet formed in the 23S intermediates. Given these considerations, we estimate that 90% of the pre-rRNA forms a duplex with the U3 snoRNA in vivo
To assess whether the chaperone complex sufficiently accelerates the rate of U3-18S duplex formation, we calculated half-lives for the reaction as a function of substrate concentration ( is based on values in ). As described in the introduction, formation of the U3-18S duplex is a prerequisite for the U3-dependent cleavages that release the 18S precursor from the pre-rRNA. In rapidly growing cells these cleavage events have an estimated half-life of ~85 s in vivo15
. The prerequisite formation of the U3-18S duplex is thus expected to be even faster. In the absence of Imp3p and Imp4p, the formation of the U3-18S duplex is not observed. In sharp contrast, in the presence of protein, the half-life for duplex formation is less than 85 s when the pre-rRNA concentration exceeds 7 nM (based on the kinetic parameters in ). This analysis supports the argument that Imp3p and Imp4p are necessary and sufficient to fulfill the need for rapid formation of this duplex in vivo
Fig. 10 The chaperone complex ensures rapid hybridization and a high duplex yield for the U3-pre-rRNA duplexes. (a) U3-18S hybridization is a pre-requisite for cleavage and release of pre-r18S, which has a half-life of 85 s in vivo15. In the presence of proteins, (more ...)
Consistent with in vivo expectations, the presence of Imp3p and Imp4p ensures a high U3-ETS duplex yield over a broad concentration range of both substrates (U3 snoRNA and pre-rRNA) based on calculated percent yield (). In the absence of protein, pre-rRNA and U3 snoRNA (assuming equimolar amounts) must exceed estimates of their nucleolar concentrations (>63 µM) to achieve high duplex yield (>90%). In contrast, lower substrate concentrations (> 0.63 µM), in line with in vivo estimates, are sufficient to ensure high U3-ETS duplex yield in the presence of Imp3p and Imp4p.
The U3-ETS and U3-18S hybridizations were modeled as separate bimolecular reactions because the pre-rRNA was divided into two minimal substrates (ETS and 18S); however, intramolecular reactions may also occur in vivo with full-length pre-rRNA (). During pre-rRNA transcription, the U3-ETS duplex may hybridize first as a bimolecular reaction because the ETS site is transcribed before the 18S site. A stable U3-ETS duplex is needed for subsequent intramolecular U3-18S hybridization. The half-life for this intramolecular reaction may occur even faster than those in due to higher effective concentration (lower entropic barrier). It is reasonable to assume that unfolding of the box A/A’ stem structure will still limit the U3-18S reaction in the absence of protein. Our in vitro studies provide evidence that the presence of Imp3p and Imp4p will alleviate this kinetic unfolding barrier to accelerate U3-18S hybridization and enhance the stability of the U3-ETS duplex.