BACKGROUND
alternative pre-mrna splicing significantly increases genome diversity with recent measurements suggesting that greater than 90% of genes undergo alternative splicing  <cit> .
the different protein isoforms generated through alternative splicing can alter function or cellular localization, or it may provide a mechanism to regulate the levels of the protein by leading to non-productive splicing and rna turnover .
regulated alternative splicing is dependent upon the alternative splicing factors present in specific cell types or at different developmental stages.
many of these alternative splicing factors appear to regulate hundreds of exons and they function by recognizing specific rna sequence elements within or near the regulated exons .
muscleblind-like  <dig>  is an alternative splicing factor that is associated with the disease myotonic dystrophy .
patients with dm have a ctg or cctg repeat expansion in an untranslated region of their genome.
for dm type  <dig> , the ctg repeats are found in the 3' utr of the dmpk gene, and for dm type  <dig> , the cctg repeats are found within intron  <dig> of the cchc-type zinc finger , nucleic acid binding protein  gene  <cit> .
these ctg and cctg repeats, when transcribed, form cug/ccug rna stem loops that bind and sequester mbnl <dig> into nuclear foci resulting in a loss of normal mbnl <dig> function in the cell .
in studying the molecular basis of dm and the role mbnl <dig> plays, it was discovered that mbnl <dig> regulates the alternative splicing of a host of developmentally regulated transcripts  <cit> .
in the cells of dm patients, adult splicing patterns revert back to embryonic splicing patterns and these incorrect splicing events are thought to cause many of the symptoms of the disease .
several mbnl <dig> binding sites have been identified, and a few of these have been characterized  <cit> .
complicating the analysis of mbnl <dig> binding to these sites and the cug and ccug repeats has been differentiating between the effects of rna structure on mbnl <dig> binding and sequence specific rna binding.
mbnl <dig> interacts with rna through its four ccch znfs, although it is possible that other regions of the protein interact with rna as well or play an important role in regulating splicing  <cit> .
the znfs of mbnl <dig> fold into two compact domains znfs1/ <dig> and znfs3/ <dig>  <cit> .
a crystal structure of znfs3/ <dig> in complex with a  <dig> mer rna  revealed that these znfs bind single-stranded rna through specific recognition of the watson-crick face of the gc dinucleotide  <cit> .
each znf  interacts with rna in a similar manner and the structure of the znfs domain in complex with rna lead teplova and patel to propose that mbnl <dig> binds rna in a looped conformation  <cit> .
this model predicts that for each znf to bind a gc dinucleotide a significant rna linker must separate the gc dinucleotides because the rna binding surfaces of znfs3/ <dig>  are on opposing sides of the domain  <cit> .
binding to rnas containing three different rna linkers of  <dig>   <dig> and  <dig> nucleotides between the gc dinucleotides to znfs3/ <dig> of mbnl <dig> supported this model  <cit> .
however these experiments were only performed with the isolated znfs3/ <dig> and it was unclear if the spacer would be important in the context of the four znfs of mbnl <dig>  to determine if the spacing between gc dinucleotides was important for mbnl <dig> binding and to determine if the sequence of the rna linker between the gc dinucleotides affected rna binding, a series of single-stranded rnas varying linker distances and different linker sequences were studied with mbnl <dig>
RESULTS
to better understand the rna binding specificity of human mbnl <dig>  we used a version of the protein that contains amino acids 2- <dig>  previous studies have shown that mbnl <dig> 2- <dig>  which contains all four znfs, binds rna as well as the corresponding full-length protein  <cit> .
this version of the protein was used due to its ease in expression and purification in a bacterial system.
this region of the protein has been shown to be sufficient for splicing regulation  <cit> .
determination of the number of gc dinucleotides required for high affinity binding by mbnl1
according to the crystal structure of the mbnl <dig> znfs3/ <dig> - rna complex  <cit> , the primary determinant of rna specificity is the recognition of the watson-crick faces of the gc dinucleotide by the znfs.
to test the importance of the gc dinucleotide in the context of single-stranded rna for binding to mbnl <dig>  one to four of these motifs were placed into a poly-uridine rna .
uridines were chosen as the template because we had previously shown that mbnl <dig> generally prefers uridines compared to the other three nucleotides  <cit> .
additionally, poly-uridine lacks rna structure so the role of rna structure in binding can be minimized allowing the focus to be on the role of specificity in a single-stranded rna substrate.
to determine if these rnas are single-stranded, a thermal denaturation assay was used to monitor the structure of these rnas.
for all rnas tested with one to four gc dinucleotides, the melting point was below 40°c , while the  <dig> rna which was previously shown to be structured and bind mbnl <dig>  <cit>  has a melting point of  <dig>  +/-  <dig> °c .
these thermal denaturation results indicate that these model rnas are primarily single-stranded and can be used to test mbnl1's specificity in the absence of rna structure.
a possibility we cannot discount is that when mbnl <dig> binds rna it could stabilize weak rna structures; therefore we cannot completely rule out the role of rna structure.
further experiments would be needed to explore this possibility.
as expected, when both gc dinucleotides are mutated to any other sequence or their polarity is reversed , mbnl <dig> binding is eliminated at the concentrations of protein tested .
although we had found previously that mbnl <dig> prefers uridines over the other three nucleotides  <cit> , these results showed that mbnl <dig> does not bind to long stretches of uridines alone with appreciable affinity.
surprisingly, the presence of a single gc dinucleotide was sufficient for high affinity binding by mbnl <dig> .
the 1gc rna bound with a kd of  <dig> nm while the 2gc rna bound with a kd of  <dig> nm.
these results indicate that mbnl <dig> requires only one gc dinucleotide for one of its four znfs to achieve significant rna binding.
although the 1gc rna bound with relatively high affinity, the complex appeared to dissociate during the gel shift assay based on the observed smearing .
to confirm the gel shift assay results, a competition assay was used.
in this assay, mbnl <dig> was pre-bound to a radioactive 2gc rna to form a stable complex.
then increasing amounts of cold competitive rna were added .
the competition results showed a larger difference between the 1gc and 2gc rnas compared to the binding assays ; however these results are consistent with the gel shift assay and further demonstrate that mbnl <dig> only requires one gc dinucleotide for nanomolar affinity binding by mbnl <dig> to rna.
the differences in relative affinities between the 2gc and 1gc rnas in the gel shift and competition assays may be due to the instability of the 1gc-mbnl complex making quantification of this complex in the gel shift assay difficult to measure accurately.
to determine if more than two gc dinucleotides increased rna binding by mbnl <dig>  a longer rna  containing up to four gc dinucleotides was used for binding studies.
the oligonucleotides with two, three, or four dinucleotides all behaved in a similar manner .
mbnl <dig> formed a single complex with all rnas and the kds were all within error of each other .
a rna containing one gc dinucleotide  was also tested in the context of the longer rna and it showed an approximately 5-fold increase in kd with lower complex stability  as seen with the shorter 1gc rna .
these results indicate that adding more than two gc dinucleotides did not enhance mbnl <dig> binding to rna in this context.
the observation that rnas containing three or four gc dinucleotides didn't bind mbnl <dig> better than rnas containing just two gc dinucleotides was surprising.
it suggests that only two of the four znfs require gc motifs for high affinity binding while the other two znfs may bind other sequences such as a poly-uridine or are involved in an intramolecular interaction that precludes rna binding.
this intramolecular interaction is supported by the mbnl <dig> znfs3/ <dig> crystal structure which formed a znf <dig> - znf <dig> interaction with a  <dig> Å <dig> interface that would preclude znf <dig> from binding rna  <cit> .
thus we might predict that znf <dig> and znf <dig> are involved in a higher order structure while znf <dig> and znf <dig> are involved in rna binding.
although it is possible that the two znfs involved in rna binding could be any of the four znfs, as none of the znfs alone have been directly tested for rna binding.
in comparing this model with the known endogenous mbnl <dig> targets, it can be seen that many of these binding sites contain only two ygcy motifs suggesting that a mode in which mbnl <dig> uses only two of its znfs to bind ygcy motifs could be a common type of interaction for mbnl <dig>  <cit> .
an alternative explanation for the lack of increased mbnl <dig> binding to more than two gc motifs is that the rna we used may not allow all four gc dinucleotides to bind all four znfs.
however, in rna 3gc# <dig> the three gc dinucleotides are spaced by nine and twelve nucleotides, and the model proposed by teplova and patel describing mbnl <dig> binding to rnas with multiple gc dinucleotides suggests that this type of spacing would allow for binding by three of the four znfs  <cit> .
our data described later also suggests that an rna containing two gc dinucleotides with only a single nucleotide spacing does not result in a dramatic decrease in binding.
this supports the idea that the spacing of the gc dinucleotides was not a problem in these rna constructs.
the role of nucleotides adjacent to the gc dinucleotides in mbnl <dig> rna binding
our previous research showed enrichment for ygcy in a selex assay of mbnl <dig> 2- <dig>  however the importance of the pyrimidines upstream and downstream of the gc was never directly tested  <cit> .
therefore we set about to determine the importance of nucleotides upstream and downstream of the gc dinucleotide by making mutations to these nucleotides in the context of the 2gc rna .
the cgcc rna bound as well as the 2gc rna, with kds of  <dig> nm and  <dig> nm respectively .
replacing all of the uridines with cytidines decreased binding by 10-fold .
however if the upstream and downstream cytidines were replaced with uridines , wild type levels of rna binding were restored.
these results suggest that mbnl <dig> requires some uridines to be near the gc dinucleotides but that there is some flexibility in their location relative to the gc motifs.
replacing the pyrimidines with purines between the gc motifs significantly reduced mbnl <dig> binding.
the presence of adenosines adjacent to the gc dinucleotide weakened binding to mbnl <dig> by 5-fold while guanosines in these positions reduced binding by 40-fold .
placing two ugcu motifs into a polya rna resulted in more than a 10-fold reduction in binding compared to the 2gc rna , indicating that positions outside of the ugcu motifs also affect mbnl1's ability to bind rna.
our characterization of mbnl <dig> binding specificity is consistent with our previously proposed mbnl <dig> binding site motif of ygcy  <cit> .
however, there seems to be an additional requirement of a uridine rich context as seen by the 10-fold decrease in binding between a cytidine rich and a uridine rich environment.
this ygcy and u-rich requirement is not explained by the crystal structure of znfs3/ <dig> in complex with rna.
in this structure znf <dig> only interacts with the gc dinucleotide and znf <dig> makes one base specific contact with the downstream uridine, therefore recognizing a gcu motif  <cit> .
one explanation for this apparent discrepancy between our results and the crystal structure is that important protein-rna contacts were not captured in the crystal structure.
it is possible that znfs1/ <dig> makes different rna contacts than znfs3/ <dig> or amino acids outside of the crystallized domains interact with rna.
further studies are required to determine which regions of the protein are responsible for these additional rna sequence requirements.
the effects of the separation between gc dinucleotides in mbnl <dig> rna binding
the crystal structures of the individual znfs domains of mbnl <dig> suggested that a rna linker of  <dig> Å would be required to allow two znfs in one domain to each bind a gc dinucleotide  <cit> .
in support of this model, teplova and patel showed that decreasing the gc dinucleotide linker from  <dig> or  <dig> nucleotides to  <dig> nucleotides reduced rna binding with the znfs3/ <dig> domain  <cit> .
to determine if the distance between the two gc dinucleotides affects the binding of mbnl <dig> containing four znfs we systematically varied the distance between the gc motifs in the 2gc rna .
interestingly, the rnas that bound mbnl <dig> with the highest affinity  had the longest and shortest linkers .
the rnas with intermediate length rna linkers of seven to  <dig> nucleotides bound mbnl <dig> with kds between  <dig> and  <dig> nm.
mbnl1's ability to bind all of these rnas with relatively high affinity suggests that the two znfs domains can adopt different orientations to recognize the gc dinucleotides.
the weaker binding to the rnas containing intermediate length rna linkers could be due to mbnl <dig> needing to adopt a slightly less favorable conformation to bind the rna compared to the conformation adopted when binding short or long linker rnas.
in mbnl <dig>  the znfs domains are separated by  <dig> amino acids and we propose that this region of the protein provides flexibility that allows the znfs domains to bind rnas with a wide range of spacing between the primary gc motifs.
figure  <dig> shows a model in which the znfs domains are oriented such that znf <dig> and znf <dig> are binding an rna with a single nucleotide spacer between the gc dinucleotides.
although znfs3/ <dig> was crystallized with a single gc rna, there are multiple rnas bound to this domain in the crystal structure.
this structure allowed us to model how mbnl <dig> might bind an rna containing two gc dinucleotides, assuming that the two rnas observed in the crystal structure would normally be found in one longer rna.
the orientation of the znfs domains shown in figure  <dig> may favor znfs domain-domain interactions that could contribute to the high affinity rna binding seen with the  <dig> and  <dig> nucleotide spacer rnas .
other orientations of the znfs domains are possible such that znf <dig> and znf <dig> bind the rna for example.
the flexibility of mbnl <dig> may explain why it is able to recognize a diverse set of rna targets that include the toxic cug/ccug repeats and many different sites within pre-mrnas.
the proposed model explains how mbnl <dig> may recognize a wide range of single-stranded rna targets, but it does not address how mbnl <dig> interacts with structured rnas such as the cug/ccug repeats or stem-loop targets found in its pre-mrna targets.
it is possible that mbnl <dig> binds the ygcy motifs in the structured rnas when these nucleotides are accessible  in these rna targets.
the flexibility of the linker between the znfs domains would allow mnbl <dig> to easily reach two sites on different parts of a structured rna.
alternatively, mbnl <dig> may recognize its structured rna targets using a different mode of recognition.
in the future biochemical approaches using techniques in addition to the gel shift assay and structural studies will be necessary to determine if mbnl <dig> use one or more modes of binding to recognize single-stranded and structured rna substrates.
CONCLUSIONS
this study suggests that the znfs domains of mbnl <dig> are linked together by a highly flexible linker , which allows mbnl <dig> to recognize a wide variety of rna targets.
although mbnl <dig> contains four znfs, it appears that only two znfs binding gc motifs are necessary for high affinity rna binding.
materials and methods
protein purification
the mbnl <dig> protein used in these studies contains amino acids 2- <dig> and was described previously  <cit> .
purification of the protein and removal of the gst tag was described previously  <cit>  except that for our studies the protein was stored at -80°c rather than at -20°c.
rna synthesis, labeling, and purification
all rnas were synthesized by integrated dna technologies and 5' end labeled with atp.
rnas were then purified on a 10% denaturing polyacrylamide gel  and eluted into te buffer .
rna was then ethanol precipitated with glycogen and resuspended in te buffer.
finally a bio-spin  <dig> column  equilibrated in te buffer was used.
rna thermal denaturation assay
<dig>  μm rna was incubated at 95°c for two minutes in  <dig> mm nacl,  <dig> mm tris  <dig> , and  <dig> mm mgcl <dig>  rna was then incubated on ice for  <dig> minutes.
the thermal denaturation analysis was performed on a carey uv/vis spectrophotometer.
the absorbance of the rna was monitored at  <dig> nm while the temperature was increased from 15-90°c.
melting temperatures were determined by calculating the derivative of the absorbance versus temperature using the cary program "thermal".
all rnas were tested three times and tm values compared.
the tm of all the rnas used in these studies were measured at below 40°c while the  <dig> rna was measured with a tm of  <dig>  +/-  <dig> °c.
gel mobility shift assay
rna was initially heated to  <dig> °c for two minutes and then placed directly onto ice.
rna was then mixed with mbnl <dig> at concentrations indicated in the figures.
the final reaction conditions were  <dig> mm nacl,  <dig> mm tris ph  <dig> ,  <dig>  mm edta,  <dig> mg/ml bsa,  <dig>  mg/ml heparin,  <dig>  mm bme,  <dig> mm dtt,  <dig> % triton x- <dig>   <dig> mm mgcl <dig>  10% glycerol,  <dig> % bromophenol blue, and  <dig>  nm rna.
the samples were incubated at room temperature for  <dig> minutes and then loaded onto a pre-chilled 6% polyacrylamide  gel running at  <dig> volts.
once the samples were loaded the voltage was increased to  <dig> volts and the gel was run for  <dig> minutes.
after running, the gel was dried and exposed overnight.
quantitation of the binding curve was described previously  <cit> .
the data was fit using the equation  where m <dig> = a fitting factor, m <dig> = nm protein concentration, and m <dig> = nm kd.
competition assay
competition assays were run under the same conditions as the gel shift assays except that the protein and radioactive rna were incubated for  <dig> minutes and then the competitive rna was added to the reaction and allowed to equilibrate for  <dig> minutes.
quantitation of the competition assays was done using kaleidagraph.
the data was fit to the equation where m <dig> = competitive rna kd, m <dig> = kd to radiolabeled rna, pt = total protein added in the reaction, and m <dig> = amount of competitive rna.
when the competitive rna and the radiolabeled rna were the same, the equation was simplified to , assuming m <dig> and m <dig> are the same.
modeling of mbnl <dig> binding rna
pdb files 3d2n and 3d2s were used to create figure  <dig>  each pdb file was opened in separate pymol windows.
3d2s contained znfs3/ <dig> bound to rna.
the structure originally contained a znfs3/ <dig> dimer bound to rna.
the extra znfs3/ <dig> was removed and the rna bound to znf <dig> was removed for the figure.
in the other window the znfs1/ <dig> structure  was oriented to place the hypothesized cytidine binding pocket towards the unbound cytidine from 3d2s.
figures were then made of each of these windows and were merged in photoshop.
the amino acid linker was then added by linking the c-terminal amino acid from znfs1/ <dig> with the n-terminal amino acid from znfs3/ <dig>
authors' contributions
dc performed most of the experiments, data analysis, and drafted the manuscript.
rh, pb, and kj performed many of the replicate binding assays.
dpg performed the thermal melt analysis.
jab conceived of the project, analyzed data and edited the manuscript.
all authors read and approved the final manuscript.
acknowledgements and funding
we thank members of the berglund lab for experimental advice and critical reading of the manuscript.
this research was supported by grants ar <dig> and ar053903-03s <dig> from nih to jab.

 alternative pre-mrna splicing significantly increases genome diversity with recent measurements suggesting that greater than 90% of genes undergo alternative splicing. the different protein isoforms generated through alternative splicing can alter function or cellular localization, or it may provide a mechanism to regulate the levels of the protein by leading to non-productive splicing and rna turnover. regulated alternative splicing is dependent upon the alternative splicing factors present in specific cell types or at different developmental stages. many of these alternative splicing factors appear to regulate hundreds of exons and they function by recognizing specific rna sequence elements within or near the regulated exons. muscleblind-like is an alternative splicing factor that is associated with the disease myotonic dystrophy. patients with dm have a ctg or cctg repeat expansion in an untranslated region of their genome. for dm type, the ctg repeats are found in the 3' utr of the dmpk gene, and for dm type, the cctg repeats are found within intron of the cchc-type zinc finger, nucleic acid binding protein gene. these ctg and cctg repeats, when transcribed, form cug/ccug rna stem loops that bind and sequester mbnl into nuclear foci resulting in a loss of normal mbnl function in the cell. in studying the molecular basis of dm and the role mbnl plays, it was discovered that mbnl regulates the alternative splicing of a host of developmentally regulated transcripts. in the cells of dm patients, adult splicing patterns revert back to embryonic splicing patterns and these incorrect splicing events are thought to cause many of the symptoms of the disease. several mbnl binding sites have been identified, and a few of these have been characterized. complicating the analysis of mbnl binding to these sites and the cug and ccug repeats has been differentiating between the effects of rna structure on mbnl binding and sequence specific rna binding. mbnl interacts with rna through its four ccch znfs, although it is possible that other regions of the protein interact with rna as well or play an important role in regulating splicing. the znfs of mbnl fold into two compact domains znfs1/ and znfs3/. a crystal structure of znfs3/ in complex with a mer rna revealed that these znfs bind single-stranded rna through specific recognition of the watson-crick face of the gc dinucleotide. each znf interacts with rna in a similar manner and the structure of the znfs domain in complex with rna lead teplova and patel to propose that mbnl binds rna in a looped conformation. this model predicts that for each znf to bind a gc dinucleotide a significant rna linker must separate the gc dinucleotides because the rna binding surfaces of znfs3/ are on opposing sides of the domain. binding to rnas containing three different rna linkers of and nucleotides between the gc dinucleotides to znfs3/ of mbnl supported this model. however these experiments were only performed with the isolated znfs3/ and it was unclear if the spacer would be important in the context of the four znfs of mbnl to determine if the spacing between gc dinucleotides was important for mbnl binding and to determine if the sequence of the rna linker between the gc dinucleotides affected rna binding, a series of single-stranded rnas varying linker distances and different linker sequences were studied with mbnl  to better understand the rna binding specificity of human mbnl we used a version of the protein that contains amino acids 2- previous studies have shown that mbnl 2- which contains all four znfs, binds rna as well as the corresponding full-length protein. this version of the protein was used due to its ease in expression and purification in a bacterial system. this region of the protein has been shown to be sufficient for splicing regulation. determination of the number of gc dinucleotides required for high affinity binding by mbnl1 according to the crystal structure of the mbnl znfs3/ - rna complex, the primary determinant of rna specificity is the recognition of the watson-crick faces of the gc dinucleotide by the znfs. to test the importance of the gc dinucleotide in the context of single-stranded rna for binding to mbnl one to four of these motifs were placed into a poly-uridine rna. uridines were chosen as the template because we had previously shown that mbnl generally prefers uridines compared to the other three nucleotides. additionally, poly-uridine lacks rna structure so the role of rna structure in binding can be minimized allowing the focus to be on the role of specificity in a single-stranded rna substrate. to determine if these rnas are single-stranded, a thermal denaturation assay was used to monitor the structure of these rnas. for all rnas tested with one to four gc dinucleotides, the melting point was below 40°c, while the rna which was previously shown to be structured and bind mbnl has a melting point of +/- °c. these thermal denaturation results indicate that these model rnas are primarily single-stranded and can be used to test mbnl1's specificity in the absence of rna structure. a possibility we cannot discount is that when mbnl binds rna it could stabilize weak rna structures; therefore we cannot completely rule out the role of rna structure. further experiments would be needed to explore this possibility. as expected, when both gc dinucleotides are mutated to any other sequence or their polarity is reversed, mbnl binding is eliminated at the concentrations of protein tested. although we had found previously that mbnl prefers uridines over the other three nucleotides, these results showed that mbnl does not bind to long stretches of uridines alone with appreciable affinity. surprisingly, the presence of a single gc dinucleotide was sufficient for high affinity binding by mbnl. the 1gc rna bound with a kd of nm while the 2gc rna bound with a kd of nm. these results indicate that mbnl requires only one gc dinucleotide for one of its four znfs to achieve significant rna binding. although the 1gc rna bound with relatively high affinity, the complex appeared to dissociate during the gel shift assay based on the observed smearing. to confirm the gel shift assay results, a competition assay was used. in this assay, mbnl was pre-bound to a radioactive 2gc rna to form a stable complex. then increasing amounts of cold competitive rna were added. the competition results showed a larger difference between the 1gc and 2gc rnas compared to the binding assays; however these results are consistent with the gel shift assay and further demonstrate that mbnl only requires one gc dinucleotide for nanomolar affinity binding by mbnl to rna. the differences in relative affinities between the 2gc and 1gc rnas in the gel shift and competition assays may be due to the instability of the 1gc-mbnl complex making quantification of this complex in the gel shift assay difficult to measure accurately. to determine if more than two gc dinucleotides increased rna binding by mbnl a longer rna containing up to four gc dinucleotides was used for binding studies. the oligonucleotides with two, three, or four dinucleotides all behaved in a similar manner. mbnl formed a single complex with all rnas and the kds were all within error of each other. a rna containing one gc dinucleotide was also tested in the context of the longer rna and it showed an approximately 5-fold increase in kd with lower complex stability as seen with the shorter 1gc rna. these results indicate that adding more than two gc dinucleotides did not enhance mbnl binding to rna in this context. the observation that rnas containing three or four gc dinucleotides didn't bind mbnl better than rnas containing just two gc dinucleotides was surprising. it suggests that only two of the four znfs require gc motifs for high affinity binding while the other two znfs may bind other sequences such as a poly-uridine or are involved in an intramolecular interaction that precludes rna binding. this intramolecular interaction is supported by the mbnl znfs3/ crystal structure which formed a znf - znf interaction with a Å interface that would preclude znf from binding rna. thus we might predict that znf and znf are involved in a higher order structure while znf and znf are involved in rna binding. although it is possible that the two znfs involved in rna binding could be any of the four znfs, as none of the znfs alone have been directly tested for rna binding. in comparing this model with the known endogenous mbnl targets, it can be seen that many of these binding sites contain only two ygcy motifs suggesting that a mode in which mbnl uses only two of its znfs to bind ygcy motifs could be a common type of interaction for mbnl. an alternative explanation for the lack of increased mbnl binding to more than two gc motifs is that the rna we used may not allow all four gc dinucleotides to bind all four znfs. however, in rna 3gc# the three gc dinucleotides are spaced by nine and twelve nucleotides, and the model proposed by teplova and patel describing mbnl binding to rnas with multiple gc dinucleotides suggests that this type of spacing would allow for binding by three of the four znfs. our data described later also suggests that an rna containing two gc dinucleotides with only a single nucleotide spacing does not result in a dramatic decrease in binding. this supports the idea that the spacing of the gc dinucleotides was not a problem in these rna constructs. the role of nucleotides adjacent to the gc dinucleotides in mbnl rna binding our previous research showed enrichment for ygcy in a selex assay of mbnl 2- however the importance of the pyrimidines upstream and downstream of the gc was never directly tested. therefore we set about to determine the importance of nucleotides upstream and downstream of the gc dinucleotide by making mutations to these nucleotides in the context of the 2gc rna. the cgcc rna bound as well as the 2gc rna, with kds of nm and nm respectively. replacing all of the uridines with cytidines decreased binding by 10-fold. however if the upstream and downstream cytidines were replaced with uridines, wild type levels of rna binding were restored. these results suggest that mbnl requires some uridines to be near the gc dinucleotides but that there is some flexibility in their location relative to the gc motifs. replacing the pyrimidines with purines between the gc motifs significantly reduced mbnl binding. the presence of adenosines adjacent to the gc dinucleotide weakened binding to mbnl by 5-fold while guanosines in these positions reduced binding by 40-fold. placing two ugcu motifs into a polya rna resulted in more than a 10-fold reduction in binding compared to the 2gc rna, indicating that positions outside of the ugcu motifs also affect mbnl1's ability to bind rna. our characterization of mbnl binding specificity is consistent with our previously proposed mbnl binding site motif of ygcy. however, there seems to be an additional requirement of a uridine rich context as seen by the 10-fold decrease in binding between a cytidine rich and a uridine rich environment. this ygcy and u-rich requirement is not explained by the crystal structure of znfs3/ in complex with rna. in this structure znf only interacts with the gc dinucleotide and znf makes one base specific contact with the downstream uridine, therefore recognizing a gcu motif. one explanation for this apparent discrepancy between our results and the crystal structure is that important protein-rna contacts were not captured in the crystal structure. it is possible that znfs1/ makes different rna contacts than znfs3/ or amino acids outside of the crystallized domains interact with rna. further studies are required to determine which regions of the protein are responsible for these additional rna sequence requirements. the effects of the separation between gc dinucleotides in mbnl rna binding the crystal structures of the individual znfs domains of mbnl suggested that a rna linker of Å would be required to allow two znfs in one domain to each bind a gc dinucleotide. in support of this model, teplova and patel showed that decreasing the gc dinucleotide linker from or nucleotides to nucleotides reduced rna binding with the znfs3/ domain. to determine if the distance between the two gc dinucleotides affects the binding of mbnl containing four znfs we systematically varied the distance between the gc motifs in the 2gc rna. interestingly, the rnas that bound mbnl with the highest affinity had the longest and shortest linkers. the rnas with intermediate length rna linkers of seven to nucleotides bound mbnl with kds between and nm. mbnl1's ability to bind all of these rnas with relatively high affinity suggests that the two znfs domains can adopt different orientations to recognize the gc dinucleotides. the weaker binding to the rnas containing intermediate length rna linkers could be due to mbnl needing to adopt a slightly less favorable conformation to bind the rna compared to the conformation adopted when binding short or long linker rnas. in mbnl the znfs domains are separated by amino acids and we propose that this region of the protein provides flexibility that allows the znfs domains to bind rnas with a wide range of spacing between the primary gc motifs. figure shows a model in which the znfs domains are oriented such that znf and znf are binding an rna with a single nucleotide spacer between the gc dinucleotides. although znfs3/ was crystallized with a single gc rna, there are multiple rnas bound to this domain in the crystal structure. this structure allowed us to model how mbnl might bind an rna containing two gc dinucleotides, assuming that the two rnas observed in the crystal structure would normally be found in one longer rna. the orientation of the znfs domains shown in figure may favor znfs domain-domain interactions that could contribute to the high affinity rna binding seen with the and nucleotide spacer rnas. other orientations of the znfs domains are possible such that znf and znf bind the rna for example. the flexibility of mbnl may explain why it is able to recognize a diverse set of rna targets that include the toxic cug/ccug repeats and many different sites within pre-mrnas. the proposed model explains how mbnl may recognize a wide range of single-stranded rna targets, but it does not address how mbnl interacts with structured rnas such as the cug/ccug repeats or stem-loop targets found in its pre-mrna targets. it is possible that mbnl binds the ygcy motifs in the structured rnas when these nucleotides are accessible in these rna targets. the flexibility of the linker between the znfs domains would allow mnbl to easily reach two sites on different parts of a structured rna. alternatively, mbnl may recognize its structured rna targets using a different mode of recognition. in the future biochemical approaches using techniques in addition to the gel shift assay and structural studies will be necessary to determine if mbnl use one or more modes of binding to recognize single-stranded and structured rna substrates.  this study suggests that the znfs domains of mbnl are linked together by a highly flexible linker, which allows mbnl to recognize a wide variety of rna targets. although mbnl contains four znfs, it appears that only two znfs binding gc motifs are necessary for high affinity rna binding. materials and methods protein purification the mbnl protein used in these studies contains amino acids 2- and was described previously. purification of the protein and removal of the gst tag was described previously except that for our studies the protein was stored at -80°c rather than at -20°c. rna synthesis, labeling, and purification all rnas were synthesized by integrated dna technologies and 5' end labeled with atp. rnas were then purified on a 10% denaturing polyacrylamide gel and eluted into te buffer. rna was then ethanol precipitated with glycogen and resuspended in te buffer. finally a bio-spin column equilibrated in te buffer was used. rna thermal denaturation assay μm rna was incubated at 95°c for two minutes in mm nacl, mm tris, and mm mgcl rna was then incubated on ice for minutes. the thermal denaturation analysis was performed on a carey uv/vis spectrophotometer. the absorbance of the rna was monitored at nm while the temperature was increased from 15-90°c. melting temperatures were determined by calculating the derivative of the absorbance versus temperature using the cary program "thermal". all rnas were tested three times and tm values compared. the tm of all the rnas used in these studies were measured at below 40°c while the rna was measured with a tm of +/- °c. gel mobility shift assay rna was initially heated to °c for two minutes and then placed directly onto ice. rna was then mixed with mbnl at concentrations indicated in the figures. the final reaction conditions were mm nacl, mm tris ph, mm edta, mg/ml bsa, mg/ml heparin, mm bme, mm dtt, % triton x- mm mgcl 10% glycerol, % bromophenol blue, and nm rna. the samples were incubated at room temperature for minutes and then loaded onto a pre-chilled 6% polyacrylamide gel running at volts. once the samples were loaded the voltage was increased to volts and the gel was run for minutes. after running, the gel was dried and exposed overnight. quantitation of the binding curve was described previously. the data was fit using the equation where m = a fitting factor, m = nm protein concentration, and m = nm kd. competition assay competition assays were run under the same conditions as the gel shift assays except that the protein and radioactive rna were incubated for minutes and then the competitive rna was added to the reaction and allowed to equilibrate for minutes. quantitation of the competition assays was done using kaleidagraph. the data was fit to the equation where m = competitive rna kd, m = kd to radiolabeled rna, pt = total protein added in the reaction, and m = amount of competitive rna. when the competitive rna and the radiolabeled rna were the same, the equation was simplified to, assuming m and m are the same. modeling of mbnl binding rna pdb files 3d2n and 3d2s were used to create figure each pdb file was opened in separate pymol windows. 3d2s contained znfs3/ bound to rna. the structure originally contained a znfs3/ dimer bound to rna. the extra znfs3/ was removed and the rna bound to znf was removed for the figure. in the other window the znfs1/ structure was oriented to place the hypothesized cytidine binding pocket towards the unbound cytidine from 3d2s. figures were then made of each of these windows and were merged in photoshop. the amino acid linker was then added by linking the c-terminal amino acid from znfs1/ with the n-terminal amino acid from znfs3/ authors' contributions dc performed most of the experiments, data analysis, and drafted the manuscript. rh, pb, and kj performed many of the replicate binding assays. dpg performed the thermal melt analysis. jab conceived of the project, analyzed data and edited the manuscript. all authors read and approved the final manuscript. acknowledgements and funding we thank members of the berglund lab for experimental advice and critical reading of the manuscript. this research was supported by grants ar and ar053903-03s from nih to jab.
we also showed that the rna sequences adjacent to the gc dinucleotides play an important role in mbnl binding with the following preference: uridines >cytidines >adenosines >guanosines. the sequestration of mbnl by expanded cug and ccug repeats is a major component in causing myotonic dystrophy. in addition to binding the structured expanded cug and ccug repeats; previous results suggested that mbnl binds single-stranded rnas containing gc dinucleotides. these results suggest that mbnl is highly flexible and able to adopt different conformations to recognize rnas with varying sequence configurations. however additional gc dinucleotides do not enhance rna binding. for high affinity binding by mbnl the distance between the two gc dinucleotides can vary from to nucleotides. these studies revealed that a single gc dinucleotide in poly-uridine is sufficient for mbnl binding and that a second gc dinucleotide confers higher affinity mbnl binding. although mbnl contains four znfs, only two znf - gc dinucleotide interactions are necessary for high affinity binding.
 muscleblind-like is an alternative splicing factor containing four ccch zinc fingers. the sequestration of mbnl by expanded cug and ccug repeats is a major component in causing myotonic dystrophy. in addition to binding the structured expanded cug and ccug repeats; previous results suggested that mbnl binds single-stranded rnas containing gc dinucleotides.  we performed a systematic analysis of mbnl binding to single-stranded rnas. these studies revealed that a single gc dinucleotide in poly-uridine is sufficient for mbnl binding and that a second gc dinucleotide confers higher affinity mbnl binding. however additional gc dinucleotides do not enhance rna binding. we also showed that the rna sequences adjacent to the gc dinucleotides play an important role in mbnl binding with the following preference: uridines >cytidines >adenosines >guanosines. for high affinity binding by mbnl the distance between the two gc dinucleotides can vary from to nucleotides.  these results suggest that mbnl is highly flexible and able to adopt different conformations to recognize rnas with varying sequence configurations. although mbnl contains four znfs, only two znf - gc dinucleotide interactions are necessary for high affinity binding. 