k - homology ( kh ) splicing regulator protein / fuse binding protein 2 ( ksrp / fbp2 ) is a multi - functional rna - binding protein that is involved in different steps of mrna metabolism . the protein was originally identified as an important factor in the alternative splicing of n1 neuronal rna ( 1 ) and subsequently shown to be important in the decay of a subset of mrnas containing au - rich stretches within their 3-untranslated regions ( utrs ) [ the so - called adenosine  uridine - rich elements , or ares ( 2 ) ] . a later study has uncovered an interaction between the rat orthologue of ksrp ( marta1 ) and the dendritic targeting element ( dte ) in map2 mrna 3-utr that is necessary to localize the mrna at the dendritic synapsis ( 3 ) . rna binding is central to the different functions of ksrp and is mediated by four kh domains that occupy the middle region of the protein ( figure 1 ) ( 4 ) . the role of ksrp in are - mediated mrna decay ( amd ) has been studied in depth . ksrp targets a subset of mrnas that contain ares within their 3-utr , recruiting the exosome and other de - adenylation factors . recruitment of these complexes results in polya shortening followed by 35 exonucleolytic digestion of the mrnas ( 2,4 ) . the comparison of the ares targeted by ksrp reveals that despite being au - rich , these elements have very different sequences ( 5 ) . further , although the role of the protein in pre - mrna splicing and mrna localization is still largely to be explored , it seems clear that , in those contexts , protein  rna binding does not involve are - like sequences ( 1,3 ) . no unifying element has been identified so far amongst the rna targets of ksrp . indeed the kh - fold can recognize a core 4 nt sequence within single - stranded nucleic acid regions ( 6 ) and the long , single - stranded rna ( ssrna ) can potentially fit several of these domains . this hypothesis is also consistent with in vitro dissociation constants of kh3 , kh4 and kh3kh4 in complex with the rna targets . the isolated kh domains bind to short au - rich sequences with kd values in the high micromolar range but when two ( or more ) domains are joined a sub - micromolar affinity is reached ( 7 ) ( i. daz - moreno et al . , submitted for publication ) . the broad range of sequences recognized by ksrp indicates that if we are to understand the rules directing ksrp rna recognition , we can not limit ourselves to analyse the details of the interaction with a single rna partner but we need to dissect the full capability of the protein to discriminate between different rna sequences . although ksrp has been shown to directly interact with the ares ( 4 ) , we have only little information on the binding specificity and affinity of the isolated kh domains of the protein for the rna . the only equilibrium data available show that the relative positioning of a 's and u 's within the tnf- are does not change significantly the binding affinity of kh3 and kh4 ( 7 ) . no equivalent data are available on the binding of the domains of ksrp to non - au - rich sequences . here we use scaffold - independent analysis ( sia ) ( 8) to dissect the capability of the four kh domains of ksrp to recognize rna . our experiments clarify that the four domains of ksrp have different sequence specificities , although they share a negative selectivity for polyc sequences . based on these results , we probe the affinity range of the different domains using nmr - monitored equilibrium binding assays and we define the role of kh3 as a high affinity rna - binding domain . in ksrp , the sequence preference of the single domains is conserved in the multi - domain constructs ( m. f. garcia - mayoral et al . , unpublished data and i. diaz - moreno et al . , unpublished data ) . the sia data on a single domain are used to discuss a general model for ksrp ksrp kh1 ( 130218 ) , kh2 ( 233305 ) , kh3 ( 323418 ) and kh4 ( 423525 ) proteins were obtained as described ( 7 ) . briefly , n - labeled proteins were expressed in escherichia coli bl21 ( de3 ) ( invitrogen corp , carlsbad , usa ) as his  gst fusion proteins and initially purified using nickel affinity chromatography according to the manufacturer 's instructions . the bulky his  gst fusion tags were then cleaved with tev protease and removed using a second nickel affinity step . the constructs were then further purified and buffer exchanged by gel filtration ( superdex 75 16/60 column , pharmacia , pharmacia corp , peapack , usa ) . protein purity ( always > 95% ) was assessed using sds  page and coomassie staining . protein quantification was achieved by a combination of spectrophotometry using predicted extinction coefficients and ninhydrin analysis of protein hydrolysates . all nmr spectra were recorded on a varian inova ( varian inc , palo alto , usa ) spectrometer operating at 800 mhz h frequency . the spectra were processed with the nmrpipe package ( 9 ) and analysed with sparky ( 10 ) . solutions of 2550 m n - labeled samples of kh1 , kh2 , kh3 and kh4 in 10 mm tris hcl buffer , 50 mm nacl , 1 mm tcep , ph 7.4 , were titrated with the relevant rna oligonucleotides . amide chemical shift changes as a function of rna / protein ratio were fitted to obtain the kd values for the complexes using in - house software as described in ( 11 ) . sia experiments were carried out and analysed as previously described ( 8) . in brief , the 16 rna pools ( 1 mol synthesis ) that allowed the analysis of the sequence preference in 4 nt positions were purchased as 5 biotinylated - hplc - purified oligos from curevac , germany and reconstituted in rnase - free 10 mm tris titrations of 25 m kh1 , kh2 , kh3 and kh4 samples with the different rna pools ( i.e. uannn , ucnnn , ugnnn , etc . ) were carried out at 27c in 10 mm tris h hsqc spectra were recorded at each step of the titration ( rna : protein ratios of 0:1 , 1:1 and 3:1 ) and averaged chemical shift differences were measured as described above for a subset ( 812 ) of the peaks affected by binding . full assignments of backbone resonances of free and bound kh domains were available from previous work ( 7 ) ( i. daz - moreno et al . , submitted for publication , i. daz - moreno et al . , unpublished data ) . analysis of the perturbed resonances confirmed that the selected residues are scattered across the protein  rna interface . the chemical shift changes observed upon addition of the 16 pools for each of the residues selected were displayed in four histograms ( figure 2b ) . to rank the differences between pools in a simple format that allows an immediate understanding of the binding preference , the four shifts displayed in each histogram for each peak were normalized to the highest value of the four . then the normalized value obtained for one peak in one titration was averaged over all the peaks for the same titration that are reported in each histogram . comparison of this final score for the 4 nt in the same position reports on the binding preference(s ) of the protein(s ) ( table 1 ) . ( a ) overlays of a downfield region from n - hsqc spectra recorded during four titrations of the kh3 domain of ksrp with the unnan , unncn unngn , unnun pools . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) , 1:1 ( green ) and 1:3 ( cyan ) protein  rna ratios . within the ensemble of resonances that shift , this is true for all of the titrations / pools , although for the highest scoring pools we observe a larger number of resonances in intermediate exchange ( 78 versus 23 ) . in order to analyse the data comparatively , we choose only peaks that are in fast exchange in all four titrations . as all the peaks shift because of the same protein  rna binding event , the exclusion of some peaks does not bias the results ( 8) . rna titrations with the non - randomized pools in the second ( left ) and third ( right ) positions . the weighted chemical shift changes ( av ) for the 1:3 protein : rna ratio are plotted versus the residue numbers and normalized to the highest value for those residues . for each residue the four values reported correspond to the unann , uncnn , ungnn , ununn ( left ) and unnan , unncn , unngn , unnun titrations ( right ) . kh3 prefers binding to a g both in position 2 and in position 3 of the target sequence . table 1.sia scores for the four kh domains of ksrpuxnnnunxnnunnxnunnnxkh1a0.660.350.290.53c0.610.360.320.51g0.730.9811u10.790.620.66+uggga / ca / ca / ca / ckh2a0.570.760.860.93c0.450.330.380.63g0.760.640.940.98u0.970.980.790.69+uua / ga / gcccc / ukh3a0.830.720.370.80c0.850.560.580.71g0.950.990.980.95u0.810.710.710.91+nggg / uncaa / ckh4a0.980.670.210.79c0.740.640.280.39g0.890.960.950.88u0.680.650.570.91+a / gggg / uc / ua / c / ua / ccdepending on the size of the ensembles used for the scoring , small variations in the sia score ( < 0.1 ) may occur . the results of each analysis have been summarized in the  +  and   lines .  +  is assigned to nucleotides with sia score within 0.1 from the top one . is assigned to the ones with sia score within 0.1 from the bottom one . +  and   the position is assigned an  n. sia assays for ksrp kh3 . ( a ) overlays of a downfield region from n - hsqc spectra recorded during four titrations of the kh3 domain of ksrp with the unnan , unncn unngn , unnun pools . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) , 1:1 ( green ) and 1:3 ( cyan ) protein  rna ratios . within the ensemble of resonances that shift , this is true for all of the titrations / pools , although for the highest scoring pools we observe a larger number of resonances in intermediate exchange ( 78 versus 23 ) . in order to analyse the data comparatively , we choose only peaks that are in fast exchange in all four titrations . as all the peaks shift because of the same protein  rna binding event , the exclusion of some peaks does not bias the results ( 8) . rna titrations with the non - randomized pools in the second ( left ) and third ( right ) positions . the weighted chemical shift changes ( av ) for the 1:3 protein : rna ratio are plotted versus the residue numbers and normalized to the highest value for those residues . for each residue the four values reported correspond to the unann , uncnn , ungnn , ununn ( left ) and unnan , unncn , unngn , unnun titrations ( right ) . kh3 prefers binding to a g both in position 2 and in position 3 of the target sequence . sia scores for the four kh domains of ksrp depending on the size of the ensembles used for the scoring , small variations in the sia score ( < 0.1 ) may occur . the results of each analysis have been summarized in the  +  and   lines .  +  is assigned to nucleotides with sia score within 0.1 from the top one . is assigned to the ones with sia score within 0.1 from the bottom one . when overlap exists between ksrp kh1 ( 130218 ) , kh2 ( 233305 ) , kh3 ( 323418 ) and kh4 ( 423525 ) proteins were obtained as described ( 7 ) . briefly , n - labeled proteins were expressed in escherichia coli bl21 ( de3 ) ( invitrogen corp , carlsbad , usa ) as his  gst fusion proteins and initially purified using nickel affinity chromatography according to the manufacturer 's instructions . the bulky his  gst fusion tags were then cleaved with tev protease and removed using a second nickel affinity step . the constructs were then further purified and buffer exchanged by gel filtration ( superdex 75 16/60 column , pharmacia , pharmacia corp , peapack , usa ) . protein purity ( always > 95% ) was assessed using sds  page and coomassie staining . protein quantification was achieved by a combination of spectrophotometry using predicted extinction coefficients and ninhydrin analysis of protein hydrolysates . all nmr spectra were recorded on a varian inova ( varian inc , palo alto , usa ) spectrometer operating at 800 mhz h frequency . the spectra were processed with the nmrpipe package ( 9 ) and analysed with sparky ( 10 ) . solutions of 2550 m n - labeled samples of kh1 , kh2 , kh3 and kh4 in 10 mm tris hcl buffer , 50 mm nacl , 1 mm tcep , ph 7.4 , were titrated with the relevant rna oligonucleotides . amide chemical shift changes as a function of rna / protein ratio were fitted to obtain the kd values for the complexes using in - house software as described in ( 11 ) . sia experiments were carried out and analysed as previously described ( 8) . in brief , the 16 rna pools ( 1 mol synthesis ) that allowed the analysis of the sequence preference in 4 nt positions were purchased as 5 biotinylated - hplc - purified oligos from curevac , germany and reconstituted in rnase - free 10 mm tris titrations of 25 m kh1 , kh2 , kh3 and kh4 samples with the different rna pools ( i.e. uannn , ucnnn , ugnnn , etc . ) were carried out at 27c in 10 mm tris h hsqc spectra were recorded at each step of the titration ( rna : protein ratios of 0:1 , 1:1 and 3:1 ) and averaged chemical shift differences were measured as described above for a subset ( 812 ) of the peaks affected by binding . full assignments of backbone resonances of free and bound kh domains were available from previous work ( 7 ) ( i. daz - moreno et al . , submitted for publication , i. daz - moreno et al . , unpublished data ) . analysis of the perturbed resonances confirmed that the selected residues are scattered across the protein  rna interface . the chemical shift changes observed upon addition of the 16 pools for each of the residues selected were displayed in four histograms ( figure 2b ) . to rank the differences between pools in a simple format that allows an immediate understanding of the binding preference , the four shifts displayed in each histogram for each peak were normalized to the highest value of the four . then the normalized value obtained for one peak in one titration was averaged over all the peaks for the same titration that are reported in each histogram . comparison of this final score for the 4 nt in the same position reports on the binding preference(s ) of the protein(s ) ( table 1 ) . ( a ) overlays of a downfield region from n - hsqc spectra recorded during four titrations of the kh3 domain of ksrp with the unnan , unncn unngn , unnun pools . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) , 1:1 ( green ) and 1:3 ( cyan ) protein  rna ratios . within the ensemble of resonances that shift , this is true for all of the titrations / pools , although for the highest scoring pools we observe a larger number of resonances in intermediate exchange ( 78 versus 23 ) . in order to analyse the data comparatively , we choose only peaks that are in fast exchange in all four titrations . as all the peaks shift because of the same protein  rna binding event , the exclusion of some peaks does not bias the results ( 8) . rna titrations with the non - randomized pools in the second ( left ) and third ( right ) positions . the weighted chemical shift changes ( av ) for the 1:3 protein : rna ratio are plotted versus the residue numbers and normalized to the highest value for those residues . for each residue the four values reported correspond to the unann , uncnn , ungnn , ununn ( left ) and unnan , unncn , unngn , unnun titrations ( right ) . kh3 prefers binding to a g both in position 2 and in position 3 of the target sequence . table 1.sia scores for the four kh domains of ksrpuxnnnunxnnunnxnunnnxkh1a0.660.350.290.53c0.610.360.320.51g0.730.9811u10.790.620.66+uggga / ca / ca / ca / ckh2a0.570.760.860.93c0.450.330.380.63g0.760.640.940.98u0.970.980.790.69+uua / ga / gcccc / ukh3a0.830.720.370.80c0.850.560.580.71g0.950.990.980.95u0.810.710.710.91+nggg / uncaa / ckh4a0.980.670.210.79c0.740.640.280.39g0.890.960.950.88u0.680.650.570.91+a / gggg / uc / ua / c / ua / ccdepending on the size of the ensembles used for the scoring , small variations in the sia score ( < 0.1 ) may occur . the results of each analysis have been summarized in the  +  and   lines .  +  is assigned to nucleotides with sia score within 0.1 from the top one . is assigned to the ones with sia score within 0.1 from the bottom one . +  and   the position is assigned an  n. sia assays for ksrp kh3 . ( a ) overlays of a downfield region from n - hsqc spectra recorded during four titrations of the kh3 domain of ksrp with the unnan , unncn unngn , unnun pools . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) , 1:1 ( green ) and 1:3 ( cyan ) protein  rna ratios . within the ensemble of resonances that shift , this is true for all of the titrations / pools , although for the highest scoring pools we observe a larger number of resonances in intermediate exchange ( 78 versus 23 ) . in order to analyse the data comparatively , we choose only peaks that are in fast exchange in all four titrations . as all the peaks shift because of the same protein  rna binding event , the exclusion of some peaks does not bias the results ( 8) . ( b ) rna titrations with the non - randomized pools in the second ( left ) and third ( right ) positions . the weighted chemical shift changes ( av ) for the 1:3 protein : rna ratio are plotted versus the residue numbers and normalized to the highest value for those residues . for each residue the four values reported correspond to the unann , uncnn , ungnn , ununn ( left ) and unnan , unncn , unngn , unnun titrations ( right ) . kh3 prefers binding to a g both in position 2 and in position 3 of the target sequence . sia scores for the four kh domains of ksrp depending on the size of the ensembles used for the scoring , small variations in the sia score ( < 0.1 ) may occur . the results of each analysis have been summarized in the  +  and   lines .  +  is assigned to nucleotides with sia score within 0.1 from the top one . is assigned to the ones with sia score within 0.1 from the bottom one . sia is a recently established method that ranks the nucleotide preference of an rna - binding domain for each position of the single - stranded target sequence ( 8) . the method is designed for domains that bind to rna with low - to - intermediate affinity and provides not just a preferred binding sequence , but a full analysis of the domain sequence specificity . the information obtained allows us to approach the problem of rna target choice from orthogonal perspectives ; that is allows us to identify not only the sequence(s ) that a domain selects for but also the ones that the same domain selects against . as the mrna is by and large coated by proteins , negative selection against specific the sia method has been validated on the well - characterized interaction between the kh3 domain of nova-1 protein and its rna target ( 8) . we now apply this method to each of the four domains of the ksrp protein . sia is based on a comparative analysis of the nmr - detected interactions between an array of short semi - randomized rna sequences and the target protein ( figure 2 ) . the sia score reflects the preference for a nucleotide in a specific position of the binding site on a 01 scale ( 8) ; comparison of this score for the four nucleotides provides an evaluation of the binding preference(s ) of the protein . for example , to understand the nucleotide preference of ksrp kh3 for the second position within the target rna , we compare the sia scores for the four quasi - randomized unann , ungnn , uncnn and ununn pools ( table 1 ) . the highest scorer is ungnn ( 0.99 ) , while the scores of the other three pools are significantly lower . this indicates that the preferred nucleotide in the second position of the kh3-binding site is a g. analysis of the sia scores in table 1 indicates that the four kh domains of ksrp have different sequence preferences and that different positions within the target sequence are responsible for specificity . in kh3 and kh4 , the most specific positions within the rna target are the two central ones , but this does not apply to kh1 and kh2 . the sia scores for kh1 , show that the domain selects negatively for a and c in all positions . this may explain why kh1 binds to au - rich sequences with an affinity significantly lower than that of the other domains . kh3 and kh4 that do bind to au - rich sequences with measurable affinity ( 7 ) , show a positive selectivity for g - rich sequences , while kh2 is the only domain that shows a preference for an au - rich rna . there is also a clear negative selection for c in most positions for all the domains . this is different from what we observed for the nova-1 kh3rna interaction : in this domain c is the favoured nucleotide in two of the four positions , consistently with selex and in vivo data ( 8) . further , the kh domains of proteins from the pcbp family ( including cp14 and hnrnps k / j ) are known to specifically target single - stranded c - rich sequences in the 3-utr of several mrnas ( 12 ) with nanomolar affinity . and even the chicken orthologue of ksrp , zbp2 , has been reported to transiently interact with the c - rich zipcode in -actin mrna ( 13 ) . therefore , the negative selectivity for c observed for the kh domains of ksrp does not reflect a structural constraint imposed by the kh - fold but a very specific feature of the ksrp protein . to validate the rank ordering of our sia data and to relate them to actual affinities ( 8) we measured the dissociation constants of the complexes formed between the different domains and a representative set of the preferred sequences indicated by sia ( table 1 ) . we also measured the affinity of the domains for an rna oligo of sequence ucccc that according to sia is selected against by all four ksrp domains . the results of these binding assays ( figure 3 ) confirm the sia data and provide a clear picture of the rna - binding capability of the four domains . we have previously reported that kh3 binds to au - rich rna sequences with a kd of 100 m , 3-times stronger than kh2 and kh4 , while kh1 binds to the same sequences with very low affinity ( 7 ) ( i. daz - moreno et al . we now show that kh3 binds to an sia - defined sequence ( ugggu ) 100-fold stronger than to canonical uauuau or uauuua elements ( figure 3a ) . in contrast , kh2 shows a binding affinity for the sia sequence ( uuuag ) which is similar ( within the error ) to the binding affinity for the au - rich elements described above . kh4 binding to the preferred sia sequence ( uaggg ) is only marginally stronger ( 2-fold ) than binding to the au - rich elements . finally , kh1 binds to the au - rich elements very weakly but discriminates effectively in favour of the sia targets ( uuggg ) ( figure 3a ) . this is consistent with the sia data described above , which show that this domain does not favour a in any of the positions of the target . further , it shows that kh1 binds to the rna target with low affinity and yet discriminates effectively in favour of the wanted sequence , a combination that may be useful in exploring the binding space within a large rna target . our binding data also confirm that the four individual domains select strongly against c - rich sequences as no measurable shift is observed during nmr titrations ( figure 4a ) . to evaluate the effect of this negative selectivity in the context of the four - domain protein , we tested ksrp rna interactions using a construct comprising all four domains ( kh1234 ) and two rnas : a polyc 25-mer and the tnf- are 25-mer . cd and emsa show that , while binding to the are sequence is very tight [ kd in the low nanomolar range ( 7 ) ] the interaction with the polyc is weak ( kd > > m ) ( data not shown ) , confirming that the kh domains of ksrp also select against multiple adjacent cs in the four - domain protein . figure 3.ksrp kh domains bind to their target sequences with very different affinities(a ) kd values ( m ) of the complexes between ksrp kh domains and selected rna oligos ( nb : no significant binding observed ) . best sequences , an au - rich sequence and one of the worse sequences for each domain , as defined by the sia scores of table 1 . below , the sequence preferences of the domains are summarized in a weblogo ( http://berkeley.edu/logo.cgi ) graphic format ( a larger letter and top position indicates a higher score ) . ( b ) binding isotherms for selected residues of kh1 ( kd , 318  46 ; top left ) , kh2 ( kd , 540  85 ; top right ) , kh3 ( kd , 1.5  1 ; bottom left ) and kh4 ( kd , 170  30 ; bottom right ) exemplify the different binding affinities of the kh domains of ksrp . the absolute values of the amide n chemical shift changes are represented as a function of the increasing rna : protein ratios . ( a ) overlays of a downfield region from n - hsqc spectra recorded during the titrations of the kh3 domain of ksrp with , respectively , the ucccc ( top ) , uauuau ( middle ) and ugggu ( bottom ) oligos . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) and to 1:1 ( green ) , and 1:4 ( purple ) protein  rna ratios . ( b ) binding isotherms of kh3 to its different rna targets exemplifies the increase of affinity as we build the consensus sequence . the curves report on the titrations of kh3 with uauuau ( grey fit , hollow circles ) , uaguau ( red fit , hollow circles ) , uaggua ( red fit , filled circles ) and ugggu ( grey fit , filled circles ) . ksrp kh domains bind to their target sequences with very different affinities(a ) kd values ( m ) of the complexes between ksrp kh domains and selected rna oligos ( nb : no significant binding observed ) . best sequences , an au - rich sequence and one of the worse sequences for each domain , as defined by the sia scores of table 1 . below , the sequence preferences of the domains are summarized in a weblogo ( http://berkeley.edu/logo.cgi ) graphic format ( a larger letter and top position indicates a higher score ) . ( b ) binding isotherms for selected residues of kh1 ( kd , 318  46 ; top left ) , kh2 ( kd , 540  85 ; top right ) , kh3 ( kd , 1.5  1 ; bottom left ) and kh4 ( kd , 170  30 ; bottom right ) exemplify the different binding affinities of the kh domains of ksrp . the absolute values of the amide n chemical shift changes are represented as a function of the increasing rna : protein ratios . ( a ) overlays of a downfield region from n - hsqc spectra recorded during the titrations of the kh3 domain of ksrp with , respectively , the ucccc ( top ) , uauuau ( middle ) and ugggu ( bottom ) oligos . each panel displays the superimposition of three spectra corresponding to the free protein ( red ) and to 1:1 ( green ) , and 1:4 ( purple ) protein  rna ratios . ( b ) binding isotherms of kh3 to its different rna targets exemplifies the increase of affinity as we build the consensus sequence . the curves report on the titrations of kh3 with uauuau ( grey fit , hollow circles ) , uaguau ( red fit , hollow circles ) , uaggua ( red fit , filled circles ) and ugggu ( grey fit , filled circles ) . it is worth mentioning that the oligo - nucleotides used in our binding assays are both 6-mer ( tnf--derived ) and 5-mers ( sia - derived ) . in principle , it is possible that the sixth nucleotide is involved in additional important contacts with the protein . however , we do not see additional chemical shift changes for a 6-mer with respect to a 5-mer indicating that a similar range of contacts is likely to take place . in fact , the largest number of shifts is observed when titrating the ugggu and uaggg 5-mers into , respectively , kh3 and kh4 and during titrations with the au - rich 6-mers . further , the sia - derived specific 5-mers bind the kh domains with higher affinity than the tnf--derived 6-mers , so any possible difference due to the sixth nucleotide would increase the gap between specific and non - specific sequence reported here . the negative specificity observed for the c - rich pentamers instead has been validated using rna 25-mers , as described above . we have previously shown that kh3 affinity for au - rich sequences is higher than that of kh2 or kh4 for the same sequences . we show here that the domain is able to recognize a g - rich sequence with micromolar kds . this affinity is equivalent to that of the isolated nova-1 kh3 for a short rna 5-mer containing its target sequence ( 8) and indicates that ksrp kh3 discriminates between specific and non - specific rna sequences with more than 100-fold efficiency . in summary , while kh1 , kh2 and kh4 bind to the favoured ssrna sequences with kds > 100 m , the kd of kh3 in complex with the specific target is 12 m . this suggests that , in targets containing g - rich sequences , kh3 defines the frame of recognition of the whole ksrp . this general model is validated by the recent discovery that kh3 binding docks ksrp to the ggg - containing apical loop of let7a mirna precursor and stimulates pri ( and pre)-mirna processing ( m. trabucchi et al . however , a three - g stretch is rare within the are targets of ksrp : the au - rich sequences bound by the protein must be in a single - stranded conformation and insertion of three consecutive gs is likely to favour base pairing with other 3-utr sequences . we therefore wondered if the recognition of g - rich sequences by ksrp was relevant mostly to the interaction with its non - are targets . sia data report on nucleotide preference in one position of the target and are largely uncoupled from the sequence context . therefore , we can expect that the insertion of isolated gs ( or gg elements ) within au - rich elements would stabilize a specific complex favouring kh3 binding . single gs do not promote the formation of stable secondary structure elements in an au - rich context and are indeed found in many of the are targets of ksrp . we examined the binding affinity of kh3 for single and double g motifs embedded within are sequences . the kh3-uaguau and uagguu complexes have kds of 26  5 and 6  2 m , respectively ( figure 4b ) . insertion of an isolated g within the tnf- are - derived au - rich sequence leads to a 5-fold increase in kh3 affinity ; insertion of a second g leads to a further 4-fold increase . this clearly shows that kh3 would recognize single and double gs in an au - rich context , and that this recognition is likely to be a general feature of the rna targets of ksrp . ksrp recognizes its rna targets with the combined action of multiple domains ( 4,7 ) and the discriminatory role of kh1 , kh2 and kh4 in rna binding is subtle but not necessarily less important than the one of kh3 . a combination of two or three of the kh domains of ksrp , including kh3 , is necessary to reach a kd in the nanomolar range , but the kh domains of ksrp show a strong negative selectivity towards a range of sequences . among these sequences polyc mrna regions within the 3-utr are expected to be single - stranded , as are au rich ones , and are targeted by multi - kh regulatory proteins such as hnrnp k and other poly - c - binding proteins . a negative selectivity for cs restricts binding of ksrp to a subset of the possible ssrna targets and contributes to the specificity of recognition . it is important to point out that ksrp can bind sequences with interspersed cs and it is only when several sequential cs are present that negative selectivity applies . it is worth mentioning that the in vitro selected rna targets of hur , an rrm - containing are - binding protein that acts antagonistically to ksrp often contain isolated gs but very rarely cs ( 14 ) . the role played by non - sequence - specific rna - binding domains is a long - standing issue in rna recognition by multi - domain proteins . it has been proposed that some multi - domain rna - binding proteins like hnrnp k use a combination of sequence - specific and non - sequence - specific domains to select their rna targets ( 15,16 ) . our data show that ksrp also uses a combination of high and low specificity domains to recognize the target and that , if a g - rich sequence is present kh3 would assume a leading role in defining the binding frame on the target rna . however , the functional binding of the rna needs further interactions and it is interesting to consider the effect of a negative selectivity by the other domains in discriminating between target and non - target rnas . as a more in - depth analysis of sequence preference in ssrna - binding proteins is undertaken , negative selectivity may be recognized as an effective answer to the problem of preventing unwanted protein  rna interactions .
k - homology ( kh ) splicing regulator protein ( ksrp ) is a multi - domain rna - binding protein that regulates different steps of mrna metabolism , from mrna splicing to mrna decay , interacting with a broad range of rna sequences . to understand how ksrp recognizes its different rna targets it is necessary to define the general rules of ksrp  rna interaction . 
 we describe here a complete scaffold - independent analysis of the rna - binding potential of the four kh domains of ksrp . 
 the analysis shows that kh3 binds to the rna with a significantly higher affinity than the other domains and recognizes specifically a g - rich target . 
 it also demonstrates that the other kh domains of ksrp display different sequence preferences explaining the broad range of targets recognized by the protein . 
 further , ksrp shows a strong negative selectivity for sequences containing several adjacent cytosines limiting the target choice of ksrp within single - stranded rna regions . 
 the in - depth analysis of the rna - binding potential of the kh domains of ksrp provides us with an understanding of the role of low sequence specificity domains in rna recognition by multi - domain rna - binding proteins .