Convergent Transmission of RNAi Guide-Target Mismatch Information across Argonaute Internal Allosteric Network
[['RNA interference ( RNAi ) is a fundamental mechanism for regulating the expression of genes in a variety of contexts .', 'It is a process by which a short dsRNA , such as a short interfering RNA ( siRNA ) or microRNA ( miRNA ) , can induce sequence-specific silencing of genes at the mRNA stage , preventing their translation into proteins .', 'The short dsRNA contributes one of its strands , the guide strand , to bind with an Argonaute protein [1] .', 'The resulting complex forms the central component in the multimeric RNA Induced Silencing Complex ( RISC ) , which hybridizes to complementary mRNAs and silences them [2] .', 'Although this is a sequence-specific process , guide and target need not be fully complementary , expanding the set of sequences that may be targeted by a single guide sequence [3] .', 'The identity and position of any mismatched base pair variably affects the specific set of target genes and the extent to which they are silenced .', 'This allows a single guide strand to potentially inactivate multiple proteins involved in multiple pathways , yielding wide-ranging effects .', 'Discrimination of targets is accomplished partly through decreased binding affinity of the mismatched target; however , the inhibition of catalysis from the bound state may also play a significant role [4] .', 'Mismatched base pairs also influence the assembly and maturation of RISC , which involves the loading of a short dsRNA into an Argonaute protein .', 'Different Argonaute subtypes require the loaded dsRNA to have mismatches in different sequence positions for the recruitment of accessory proteins to form the finished RISC [5] .', 'Correctly modifying the relative affinities of certain components of RISC for successful assembly hence depends on the ability of these accessory proteins to sense the presence of these mismatches .', 'Both of these functional aspects of Argonaute involve the remote sensing of a structural perturbation .', 'In particular , a mismatched base pair must somehow inform other regions of the structure of its presence , producing the endpoint effect of modification of silencing activity .', 'Information transfer of this nature is commonplace in biomacromolecules .', 'It may take the form of a redistribution of the inherent conformational-energetic equilibrium of the structure: enhanced deformability of nucleic acids has been suggested to be a dynamical signal for sequence recognition by proteins [6] , [7] , mutations in aspartate transcarbamoylase produce distinct states in the conformational equilibrium [8] , [9] , and ligand binding to hen egg white lysozyme ( HEWL ) biases the conformational distribution as sampled computationally , stabilizing a particular helix , suggesting long range communication between the ligand and the helix [10] .', 'In this work , we examine this intramolecular signal transduction by studying the Argonaute ternary complex , composed of an Argonaute protein bound to a guide-target nucleic acid hybrid .', 'We examine the interaction correlation network as derived from molecular dynamics simulations of the Thermus thermophilus Argonaute ternary complex and determine the effect of introducing a single guide-target mismatch in each of several positions .', 'Our methods relate residue dynamical couplings to residue energetic couplings , viewing the influence of the perturbing factor under question as a pathway that emanates from the site of perturbation and propagates information among different regions of the same structure [11] .', 'Pathway approaches in general have the advantage that local correlations in atomic positions are easily observable on relatively short timescales such as those accessible through molecular dynamics simulation .', '( Relationships among residues may also be derived from nonphysical data: one method , operating on the assumption that coupled residue pairs are likely to be evolutionarily conserved , identifies groups of conserved residues and derives allosteric pathways from this information [12] , [13] . )', 'Using molecular dynamics simulation , we can study the atomic-level energetic fluctuations of the structure itself .', 'We employ equal-time correlations of interaction energy fluctuations — the correlation between two sets of nonbonded interaction energies between pairs of amino acid residues — in order to capture the energetic basis of internal allosteric information transmission in Argonaute [14] , [15] .', 'Interaction energy correlations among residue pairs are mapped onto the constituent residues , and groups of residues defined by high correlation values with each other are regarded to comprise allosteric pathways .', 'Previously , pathways have been identified using various operations on the residue correlation matrix , such as simple clustering [15] and graph theoretic methods like clique identification and shortest paths analysis [16] .', "Rhodopsin's conformational change upon photoactivation [17] was shown to be mediated by such an internal signaling pathway [14] .", 'Ligand binding to PDZ2 causes a dynamical change that results in modification of its residue energetic couplings [15] , which has been correlated experimentally by demonstration of changes in binding partner selectivity related to single residue mutations [18] .', 'Our work builds on these previous efforts , which were successful for smaller proteins .', 'However , in tightly packed proteins , as the number of residues N increases , the number of significant residue-residue interactions is proportional to N2 .', 'This causes the number of significant correlations to increase substantially , making it difficult to identify distinct coherent narrow internal signaling pathways in large , tightly packed proteins such as Argonaute .', 'In the present work , rather than identifying specific pathways , we instead determine the overall directionality of the relevant pathways as well as identifying specific endpoints .', 'In this way , we provide evidence for a dynamical link between mismatched base pairs and Argonaute residues on the surface and near or in the active site .', 'Even though the multimeric composition and organization of RISC is not presently known , a study of how Argonaute — as the central component of RISC from which an allosteric signal must originate — responds to mismatches in its nucleic-acid-containing complex can provide insight into these allosteric effects .', 'We propose a model of internal allostery in which transmission of information is mediated by a distributed set of intermediate residues , such that a small number of key residues are perturbed by any of several different small structural perturbations .', 'We undertake a study that does not have a convenient experimental equivalent , in order to better inform present and future experimental models .'], ['Examining the residue correlation matrix for the TtAgo ternary complex shows that there is a widely distributed set of correlations among residues in all regions of the protein .', 'This is shown in Figure 1a: note the widely distributed regions of high correlation values .', 'This represents an organized set of interaction pathways , described by energetic couplings , that spans the structure .', 'Residues that are part of the same pathway would have high correlation values with each other .', 'However , specific pathways do not present themselves as an obvious property of the matrix .', 'Previous workers had identified pathways by regarding the residue correlation matrix as a similarity matrix and using clustering methods to identify groups of residues associated by large correlation values with each other .', 'We repeated this approach , using the Markov Cluster algorithm ( MCL ) [19] , as previously used with a smaller protein [15] , and subsequently , average-linkage divisive hierarchical clustering [20] .', 'However , with both methods , as the clustering granularity was progressively decreased , large numbers of singleton clusters were resolved .', 'These methods did not identify a pathway ( or pathways ) but rather suggested that in the setting of a residue correlation matrix densely populated with high similarity values , there are many interaction correlation pathways that are not clearly distinct from one another .', 'This is consistent with the fact that Argonaute is a large protein with a large number of residue-residue interactions , and highlights the difficulty of isolating individual internal allosteric pathways in proteins of this large size .', 'We next took a different approach to identifying pathway structure , employing a different similarity metric while retaining the use of average-linkage divisive hierarchical clustering .', 'In this approach , the residue correlation matrix is treated as a matrix of observations by residue , where each “observation” for a given residue represents the degree of correlation with another particular residue .', 'Starting with one large cluster containing all residues , each cluster is then recursively divided by choosing the cluster with the greatest internal dissimilarity at each step .', 'In this way , a given pair of residues would only be assigned to the same cluster if they both correlated strongly to similar subsets of residues .', 'Importantly , this pair of residues need not actually have a strong correlation with each other .', 'With this metric , the meaning of a given cluster is that it is a “slice” across a set of correlation pathways rather than a discrete pathway .', 'This is illustrated schematically in Figure 2a , where each circle represents a residue and each connecting line represents a high correlation value between two residues .', 'Note that the red residues at the bottom of the diagram are strongly connected to the blue residues , but are not connected to each other .', 'Hence they are grouped together .', 'Similarly , the blue residues connect strongly to the red residues and the green residues , and as such are grouped together .', 'Finally , the green residues connect primarily to the blue residues and are grouped together .', 'Note that clustering in this manner does not resolve individual correlation pathways , yet the overall directionality of the network is evident .', 'Although the specific clusters chosen are to some degree dependent on the clustering method and as such the set of correlations evident in the partitioning is not necessarily exhaustive , this clustering provides substantial insight into the overall organization governing transfer of information within the interaction correlation network .', 'Applying this concept to partitioning the residue correlation matrix for each structure , we produced a set of cross-sections of the interaction correlation network in the Argonaute ternary complex , which revealed the overall architecture of the network .', 'In all matched and mismatched ternary complexes , we found that there were roughly six major clusters of residues that were robustly prominent at a variety of clustering levels .', 'These clusters were organized in layers emanating from the active site region ( i . e . corresponding to the red cluster in Figure 2a ) , in an “onion-skin” type architecture ( see Figure 2b–d ) .', 'The largest cluster included the cores of all protein domains ( blue cluster ) , showing that they are the common link in all emanating pathways .', 'This cluster was surrounded by a distributed cluster ( green cluster ) consisting of residues on the protein surface , with which it directly interacts .', 'The central clusters ( red ) include the active site and the target scissile bond .', 'This architecture allows dynamical signals to propagate from the active site region ( red ) , through the protein domain cores ( blue ) , onward to the surface of the complex ( green ) , and vice versa .', 'This widely-distributed but non-uniform network of interaction pathways follows from the architecture of Argonaute because all domains , although structurally well defined , are in relatively close proximity to each other and therefore their nonbonded interaction energies tend to be well correlated .', 'Such architecture allows the dynamics of any given region of the complex to influence all other regions of the complex .', 'This would allow Argonaute to support allosteric interactions that would have distant endpoint effects at many regions across its surface .', 'We also examined certain individual elements of the interaction correlation network .', 'Since the guide strand seed region has been implicated in RNAi sequence selectivity , we hypothesized that in a ternary complex , the degree to which a given guide strand nucleotide can influence Argonaute protein residues would depend on its position .', 'The rationale for such a hypothesis is based on a structural argument related to the nature of nucleic acid protein interactions .', 'In the ternary complex , as one progresses along the nucleic acid sequence ( e . g . , from position 3 towards position 11 near the active site ) the base pairs are not only translated from the anchoring site towards the active site , but they also change their orientation with respect to the protein because of the helical nature of the nucleic acid .', 'In approximately 5 base pair steps , the orientation turns ∼180° , such that if the guide strand was proximal to the protein , after 5 base pairs , the target strand is now in an equivalent , albeit translated , position .', 'To some extent this was the origin of sequence-position-dependent changes in the free energy of introducing a mismatch , as was observed in our previous work [21] .', 'To probe this coupling , we calculated the correlation factor of each nucleotide with the protein as detailed in the Methods section .', 'Essentially , this is the sum total of correlation values of the nucleotide with the rest of the protein , providing a description of the degree to which interaction pathways connect each individual nucleotide with the protein .', 'In all TtAgo ternary complexes , guide strand positions 4–6 ( part of the seed region ) have increased correlation with the protein regardless of the presence of a mismatch .', 'The increased seed region correlation suggests that while structural distortion is minimal when there is a guide-target mismatch in the seed region , it has the potential to propagate a signal to the protein to a greater degree than a mismatch in the surrounding positions .', 'We also probed the dependence of the correlation factor on distance from the nucleotide ( see Figure 3 ) .', 'Interestingly , active site nucleotides ( the scissile bond is between nucleotides 10 and 11 ) had a large correlation factor with respect to nearby protein residues , but this is greatly diminished with distance , indicating that perturbations in these residues have primarily local effects .', 'By contrast , seed region nucleotides had a prominent effect in distant protein residues ( 30–50 Å ) .', 'This is congruent with existing theories stating that the seed region is the primary determinant of target selectivity , because a seed region mismatch is better able to influence the rest of the structure than a non-seed-region mismatch .', 'This is also congruent with the hypothesis that this binding free energy penalty is the primary mechanism through which mismatches in the seed region reduce RNAi cleavage activity , consistent with the results of experiments showing a decrease in RNAi catalytic efficiency due to seed region mismatches [4] , [22] .', 'Previously , we found in simulations that a guide-target mismatch tends to destabilize the ternary complex relative to the binary complex , which would disfavor target binding [21] .', 'Examining the simulation trajectories of all mismatched TtAgo ternary complexes showed that in each case , significant structural distortion was limited to the mismatched base pairs themselves and their close neighborhood .', 'In addition , for all simulations of the mismatched ternary complexes , we observed that the RMSD was less than 3 Å with respect to the fully-matched ternary complex .', 'This suggests that the dynamical perturbations emanating from the mismatch site do not cause a large structural change even though their energetic change is quite substantial and highly significant with respect to function .', 'We wished to determine how this energetic change affected the interaction correlation network .', 'To do so , we compared the residue correlation matrix of the fully-matched TtAgo ternary complex with those of mismatched TtAgo ternary complexes .', 'To determine whether gross interaction pathway reorganization took place within the protein , we designed and used a similarity metric designed to describe the degree to which the general pattern of internal correlation pathways is preserved when a mismatch is introduced .', 'The method is described in detail in the Methods section , and is briefly described below .', 'We were primarily interested in the degree to which strong correlations were preserved or lost across the two structures in a given comparison , so we excluded from consideration all but the strong correlations .', 'In order to identify these strong correlations for a given residue correlation matrix , we plotted all correlation values , extracted from the matrix , in ascending order .', 'This resulted in a curve with two distinct regions — a low-valued region representing small correlations and a high-valued region representing strong correlations ( see Supporting Figure S2 ) .', 'In all cases , the high-valued region represented the top 7 . 4% of values , which for a given residue correlation matrix was approximately 17350 distinct correlation values , out of ( N2−N ) /2\u200a=\u200a234270 possible ( given that the residue correlation matrix is symmetric ) .', 'To compare two residue correlation matrices A and B , each cell at Aij was examined along with its corresponding cell Bij to produce a combined similarity score ( see Methods ) .', 'Each corresponding pair where both had strong correlations in the top 7 . 4% subset contributed a 1 and those correlations that were unmatched contributed a zero .', 'Those correlations that were not in the top 7 . 4% subset were excluded .', 'The similarity score was therefore the fraction of the corresponding pairs out of the total number of correlations preserved in the top 7 . 4% .', 'The intuition underlying this method is that in the case of full preservation of the interaction correlation network , if there is a strong correlation between residues i and j in a fully-matched structure , then i and j should also have a strong correlation in the mismatched structure , and vice versa .', 'Each score was compared to an expected similarity score between randomized versions of the same two matrices in order to assess for significance ( see Methods ) .', 'In all cases only the Argonaute protein was included in the comparison .', 'Tables 1 and 2 show comparisons using the similarity score between various matched and mismatched ternary complexes .', 'Every 0 . 001 increment in the similarity score represents approximately 35 modified strong interactions .', 'The maximum possible similarity score is 1 , indicating a fully-preserved allosteric network architecture that would preserve the dynamics of surface residues interacting with allosteric partners .', 'The similarity scores between matched and mismatched ternary complexes are in the range 0 . 9904–0 . 9911 , which implies that there were on the order of 300 modified strong correlations in each case .', 'Comparing the mismatched ternary complex residue correlation matrices to each other resulted in similarity scores ranging from 0 . 9977 to 0 . 9987 ( see Table 2 ) .', 'This indicates that the different mismatched ternary complexes are more similar to each other than they are to the fully-matched ternary complex .', 'For all comparisons the similarity scores were substantially higher than the randomized value of approximately 0 . 1514 , indicating that they were significant .', 'These high similarity scores suggest that the architecture of the network is quite resilient to perturbations , whether they originate from a binding event , a conformational change or the introduction of a mismatch in the nucleic acid sequence .', 'We were most interested in the ∼1% of variability in the network arising from a guide-target mismatch .', 'Since the only structural difference between matched and mismatched ternary complexes was within the mismatched base pair itself , this dissimilarity can be considered to arise from the presence of the mismatched base pair .', 'We therefore designed an approach to identify specific residues affected by these structural perturbations .', 'We observed that a single mismatch can modify the entire interaction correlation network , causing a subset of residues to be either coupled or decoupled from the network .', 'Given the pattern of similarity scores , we hypothesized that this subset would have significant commonality across mismatched ternary structures .', 'To identify these common elements , we examined each difference matrix resulting from element-wise subtraction of the fully-matched residue correlation matrix from each mismatched residue correlation matrix .', 'To facilitate comparisons among different matrices , each residue correlation matrix was first converted to units of standard deviations from its element mean , and only the Argonaute protein was included in these calculations .', 'For each residue correlation matrix Mmismatched corresponding to a given mismatched ternary structure , we calculated ΔM\u200a=\u200aMmismatched−Mmatched .', 'Therefore , each cell in the difference matrix ΔMij describes the change in correlation value between residues i and j due to the presence of a mismatch .', 'For a given residue pair , a gain in correlation would suggest that new interaction pathways connected to the perturbation pass through this coupling , and a loss would suggest that such pathways were removed .', 'In both cases , this means that the dynamics of a residue pair are modified in response to the perturbation .', 'However , we were interested in the average magnitudes of differences rather than their signs , so we calculated the root mean square of each cell ΔMij across all difference matrices .', 'This resulted in an averaged difference matrix ΔMrms .', 'To quantitate the degree of involvement of each residue in the modification of the residue correlation matrix due to the introduction of any of the tested mismatches , we summed the values in each column of ΔMrms to produce a vector where the ith element describes the amount of change in correlation value for residue i combined across other residues in the structure .', 'The largest values in this vector corresponded to residues which had their coupling to the interaction correlation network most affected by the mismatches .', 'Importantly , a large value could result from both increases and decreases in correlation value .', 'To determine the most important residues by this measure , we identified the 65 elements of this vector that were more than two standard deviations away from the mean of all 685 elements in the vector , and the corresponding residues were deemed the sensor residues .', 'These sensor residues gain or lose coupling with the rest of the structure in the presence of a seed region guide-target mismatch .', 'The sensor residues are listed in Table 3 and diagrammed in Figure 4 .', 'Sensor residues were found in N-terminal , PAZ , and PIWI domains as well as the linker regions , but none were found in the Mid domain .', 'Since the Mid-PIWI interface binds the 5′ end of the guide strand , a short distance from the seed region , this shows that the distribution of sensor residues was nonuniform .', 'Nearly all sensor residues were charged , with a predominance of arginine and glutamic acid residues , as well as several aspartic acid and lysine residues .', 'Since the interaction energies are dominated by electrostatics , the fact that so many sensor residues were charged is therefore not surprising .', 'Some sensor residues formed salt bridges , which could in principle change orientation , serving as an allosteric mechanism; these are listed in Table 3 .', 'The sensor residues could be divided into three categories: two residues forming part of the catalytic triad , binding groove residues , and surface residues .', 'The active site residues D546 and D660 , which interact with both Mg2+ ions in the active site , were found to be sensor residues .', 'This suggests an effect of seed region mismatches directly upon the target cleavage reaction .', 'It is likely that the mechanism of target cleavage in Argonaute is similar to that of RNase H given the structural similarities between the respective active sites — indeed , we placed the Mg2+ ions in the system by analogy with RNase H ( see Methods ) .', 'In the RNase H mechanism , an Mg2+ ion is likely to catalyze the nucleophilic attack of the scissile phosphate by a water , forming an intermediate which is bound to the other Mg2+ ion .', 'Protonation of the leaving group by an active site Asp residue completes the reaction .', 'During the course of the reaction , the inter-Mg2+ distance is modified , suggesting that the motion of these ions is significant [23] .', 'Disrupting any of these components , whether an Asp itself or an Mg2+ complexed to the Asp residues , could potentially modify the catalytic rate .', 'In addition , the binding groove sensor residues , described below , may be part of internal allosteric pathways terminating at D546 and D660 .', 'The identification of these residues strongly suggests a focused effect on the active site region due to seed region guide-target mismatches .', 'Interestingly , much of the length of the Argonaute binding groove was composed of sensor residues , demonstrating a long-range effect of seed region mismatches upon the binding groove .', 'This included a subset of residues that were located proximal to the active site; these were K191 , R192 , R194 , E203 , D246 , K248 , D249 , and E268; this region is illustrated in Figure 4a .', 'Several binding groove sensor residues were part of the PIWI domain: R482 , R574 , K575 , K618 , D546 , R548 , E597 , D598 , D660 , R661 , E665 , and R685 , also illustrated in Figure 4a .', 'Even though we employed a TtAgo guide-target structure in which both strands of the bound nucleic acid were truncated after guide strand position 12 , there were a number of sensor residues in the regions of the binding groove that would normally bind the missing nucleotides .', 'This suggests that the entire binding groove — both guide- and target-binding regions — is dynamically coupled to the seed region , and that a seed region guide-target mismatch may have an effect upon the interface between protein and nucleic acid , even in regions relatively distant from the site of the mismatch .', 'In order to estimate more accurately how the binding groove sensor residues were positioned relative to the “missing” nucleotides , we identified the sensor residues on a TtAgo ternary structure in which the missing nucleotides were resolved , but for which no experimental data regarding target cleavage as a function of mismatch is available [24] ( PDB: 3HK2 ) .', 'Binding groove sensor residues were found to be in close relationship to the “missing” nucleotides ( see Figure 4b ) .', 'Notably , near the 5′ end of the target RNA , there is a strip of sensor residues which follow the putative path of a longer target RNA as it would extend into the TtAgo protein .', 'This lends credence to the idea that the Argonaute-target interface is modified along its entire length by an internal allosteric mechanism originating at seed region guide-target mismatches .', 'Although the location of binding of RISC accessory proteins to Argonaute is not known , sensor residues on the surface of Argonaute may mediate the passage of the internal allosteric signal arising from the mismatched base pair to the surface of the protein , and by virtue of their physical accessibility to external molecules would represent potential external allosteric interfaces .', 'We wished to identify these sensor residues because they may mediate the recognition of mismatches by external proteins , such as Dicer or accessory proteins in RISC .', 'We defined a surface sensor residue as having relatively large solvent-accessible surface area ( >70 Å2 ) , as calculated using a standard probe radius of 1 . 4 Å , without being located in the binding groove .', '( The solvent-accessible surface area values are given in Table 3 and diagrammed in Supporting Figure S3 . )', 'These residues were E76 , R289 , R335 , R340 , E517 , and D521 .', 'There were relatively few regions containing such surface sensor residues .', 'Surface sensor residues R335 and R340 are located in the PIWI domain , on the opposite side of the protein from the binding groove .', 'E517 and D521 are located adjacent to each other in the PIWI domain .', 'R289 is located near the 3′ end of guide strand .', 'E76 is in the N-terminal domain .', 'In general , the locations of the sensor residues are congruent with our description of the “onion skin” architecture of the internal allosteric network with the active site at the center: the sensor residues near the active site are part of the central clusters , and those on the surface are part of peripheral clusters .', 'The overall picture is of three general categories of sensor residues with apparent functional importance: residues on the surface of the protein , which can transmit a signal to external binding partners; residues in the binding groove , which can modify the interface along the length of the bound nucleic acid distant from the seed region mismatch; and residues near and in the active site , which could potentially affect target cleavage .'], ['We present here evidence that internal allosteric signaling pathways propagate diffusely in Argonaute but can converge to produce focused distal allosteric effects , mediated through the aggregation of many small changes .', 'Methodologically , we show that key residues in internal signaling pathways can be identified by their dynamical response to a structural perturbation such as a guide-target mismatch .', 'Functionally , we show that in TtAgo these residues are located in mechanistically important regions .', 'Prior applications of the interaction correlation method have generally been with comparatively small structures such as PDZ2 [15] and pyrrolsyl-tRNA synthetase [16] , in which discrete internal allosteric pathways were identified , progressing from origin to endpoint along a narrow pathway .', 'Our analysis of Argonaute , a considerably larger protein , is consistent with a description of internal allosterics that is different but complementary — that a coherent and consistent signal acting on a small set of residues is propagated through a wide cross-section of residues which converges on these residues rather than through a small set of narrow pathways .', 'We found that the general architecture of the internal allosteric network is preserved in structures with guide-target mismatches , but that a number of key residues were consistently affected even by different mismatches .', 'This demonstrates a convergent common pathway with specific functionally relevant endpoints in Argonaute , which is a novel finding .', 'In particular , the identification of catalytic residues D546 and D660 as sensor residues suggests the existence of an internal allosteric link between seed region mismatches and the mechanism of target cleavage .', 'This result is consistent with experimental evidence; it is well known that guide-target mismatches can impair silencing activity [4] , [24] .', 'Although seed region mismatches were shown experimentally to primarily affect the binding of the target strand ( i . e . by modifying the Michaelis-Menten parameter Km ) , there was still some effect on the rate constant [4] , compatible with the hypothesis that when the mismatched ternary complex forms , target cleavage is impaired .', 'This pathway is a potential explanation for this behavior , and the first suggestion of an internal allosteric link between seed region mismatches and the active site region .', 'The effect upon the binding groove was pronounced .', 'Importantly , the contact regions for both the guide and target strands were affected .', 'We show here that the dynamics of the interface between Argonaute and bound nucleic acid can be modified by a seed region
['Introduction', 'Results', 'Discussion', 'Methods']
['In RNA interference , a guide strand derived from a short dsRNA such as a microRNA ( miRNA ) is loaded into Argonaute , the central protein in the RNA Induced Silencing Complex ( RISC ) that silences messenger RNAs on a sequence-specific basis .', 'The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used .', 'Subsequently , the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target .', 'Mismatches between guide strand and the target mRNA decrease cleavage efficiency .', 'Thus , loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites .', 'These effector sites include the active site , to prevent target cleavage; the binding groove , to modify nucleic acid binding affinity; and surface allosteric sites , to control recruitment of additional proteins to form the RISC .', 'To examine how such signals may be propagated , we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions .', 'The emerging network can be described as a set of pathways emanating from the core of the protein near the active site , distributed into the bulk of the protein , and converging upon a distributed cluster of surface residues .', 'Nucleotides in the guide strand “seed region” have a stronger relationship with the protein than other nucleotides , concordant with their importance in sequence selectivity .', 'Finally , any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein .', 'This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues .', 'These residues are in effector sites: the active site , binding groove , and surface , implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of a large number of internal allosteric pathways .']
['Cells use the process of RNA interference ( RNAi ) to help orchestrate the production of the specific set of proteins needed at a given time .', "A given messenger RNA ( mRNA ) is produced using a gene in the cell's genomic DNA as a template .", 'Each mRNA molecule is a blueprint for a particular protein and can then be “translated” into this protein .', 'Prevention of translation is called “silencing” .', 'An mRNA is chosen for silencing based on its sequence , through binding to a complementary “guide strand” which is itself bound to an Argonaute protein .', 'Some Argonaute proteins have the ability to cleave the mRNA , silencing it .', 'When the guide strand is not perfectly complementary to the target mRNA , silencing can still occur , albeit usually at a greatly reduced rate .', 'Through molecular dynamics simulation and related methods , we examine potential explanations for this behavior with respect to the movements of the atoms comprising Argonaute .', 'We show that there is a novel structural pathway through which a mismatch between guide strand and target mRNA can selectively affect certain functionally important parts of the Argonaute molecule , furthering our understanding of RNAi .']
['rna', 'rna interference', 'molecular dynamics', 'nucleic acids', 'chemistry', 'biology', 'computational chemistry', 'biophysics']