mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .
sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression
the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .
frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .
the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .
the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .
the resin was applied to a gravity flow column and washed extensively with wash buffer .
the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .
dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .
a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .
fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .
fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .
mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers .
we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .
crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .
crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 .
x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .
we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .
the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .
phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .
phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .
the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .
the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .
the resulting phases were of sufficient quality to manually build protein and rna in coot .
although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .
this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .
one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .
crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .
the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .
the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .
the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain
a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .
weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .
all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc . all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .
the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .
a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .
quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .
the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .
the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .
briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .
constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .
the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water .
a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .
we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .
a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .
the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .
all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration .
mouse dis3l2 was expressed in sf9 cells as an n - terminal strep - sumo - tev fusion protein from the pfl vector of the multibac baculovirus expression system .
sf9 cells were infected with baculovirus in hyclone ccm3 media ( thermo scientific ) at 27c . following 60 hours of expression
the cells were centrifuged at 1200 rpm and resuspended in wash buffer ( 50 mm tris ph 8 , 100 mm nacl , and 5 mm dtt ) , flash frozen in liquid nitrogen and stored at 80c .
frozen cells were thawed , nacl concentration increased to 500 mm , and then lysed by sonication .
the lysate was treated with 0.2% poly - ethylene imine ( pei ) to precipitate nucleic acids prior to ultracentrifugation at 35,000 rpm at 4c for 1 hour .
the soluble fraction was incubated with 1 ml of strep - tactin superflow resin ( iba biotagnology ) per 10 ml of lysate for 1 hour on a rolling shaker .
the resin was applied to a gravity flow column and washed extensively with wash buffer .
the cleaved protein was diluted with an equal volume of heparin buffer a ( 25 mm hepes ph 7.5 , 5 mm dtt ) to a final nacl concentration of 50 mm .
dis3l2 was loaded onto a hitrap heparin hp column ( ge life science ) equilibrated with 25 mm hepes ph 7.5 , 50 mm nacl and 5 mm dtt .
a linear gradient between 0.05 m and 1 m nacl was used to elute dis3l2 at 0.25 m nacl .
fractions that contained dis3l2 were analyzed by sds - page , pooled and concentrated to 2 ml and loaded onto a hiload 16/60 superdex 200 gel filtration column equilibrated with 10 mm tris ph 8 , 100 mm nacl , 2 mm mgcl2 and 5 mm dtt .
fractions containing dis3l2 were pooled and concentrated to 25 mg / ml , flash frozen in liquid nitrogen and stored at 80c .
mutants of dis3l2 were constructed by sequence and ligation - independent cloning ( slic ) using mutant primers .
we expressed and purified a d389n active site mutant lacking the protease - sensitive loop within csd1 ( residues 148169 and 194221 ) , and both the n ( 136 ) and c ( 857870 ) termini ( extended data figure 2 ) . the truncated dis3l2 was incubated with a 1.2 molar excess of u13 rna for 30 minutes at 20c .
crystallization was carried out by the hanging drop vapor diffusion method by mixing the dis3l2-u13 complex at 15 mg / ml with an equal volume of 100 mm tri - ammonium citrate ph 5.5 , 100200 mm ammonium chloride and 2022% peg 3350 .
crystals appeared in 12 days at 18c and were placed directly in a freshly prepared solution composed of the crystallization condition supplemented with 20% glycerol and flash frozen in liquid n2 .
x - ray diffraction data were collected to 2.95- resolution at the x25 beamline at the national synchrotron light source ( nsls ) at brookhaven national laboratory ( bnl ) ( extended data table 1 ) .
we generated a molecular replacement ( mr ) search model by pruning yeast rrp44 ( pdb 2vnu ) with the sculptor utility .
the pruned model was separated into its individual csd1 , csd2 , rnb and s1 domains .
phasing was determined by mr with phaser , using each of the domains of yeast rrp44 as individual search models .
phaser found solutions for csd2 , rnb and s1 domains , but failed to find a unique solution for csd1 .
the initial mr phases were input into the autobuild routine in phenix , where the resolve and buccaneer option for density modification and model building , respectively , was critical for phase improvement and interpretation of initial electron density maps .
the csd1 of yrrp44 was placed manually into the initial map and the autobuild routine repeated .
the resulting phases were of sufficient quality to manually build protein and rna in coot .
although a u13 homo - oligonucleotide was used in crystallization , interpretable electron density accounted for 14 nucleotides .
this is readily explained by slipping of the homo - oligou into two conformations along the rna binding cleft .
one conformation is fully threaded into the active site ( at the 0 position ) , and a second conformation occupies the 1 position .
crystallographically , these two states are indistinguishable , and thus is best accounted for by modeling 14 nucleotides .
the final model includes two copies of truncated dis3l2 with the exception of disordered residues ( 3748 , 119228 and 252258 ) , two u14 molecules , two mg ions and 6 water molecules .
the structure was refined using automatically determined ncs restraints , tls and isotropic b - factor refinement as implemented in phenix to an r / rfree of 21/25 % .
the final model was validated with molprobity , where 96.8 % of residues reside in the favored and 3.13 % in the allowed regions of the ramachandran plot ( extended data table 1 ) . the copy represented by chain
a ( dis3l2 ) and chain c ( u14 ) had appreciably better electron density and was used for all structural analysis described in this manuscript .
weak electron density , presumably due to disorder in the region surrounding u5 , precluded accurate modeling of the position of this nucleotide , so we carefully avoided over interpretation of the interactions observed in this region , though we are confident of its presence and general location .
all structural figures were generated with the pymol molecular graphics system , version 1.6 schrdinger , llc .
all exonuclease assays were conducted under multiple turnover conditions , where we measured the linear initial rate of rna degradation and calculated the specific activity ( nt degraded minute molecule of enzyme ) .
the indicated amount of dis3l2 was incubated at 30c with 100 nm 5 ' radiolabeled rna substrate in reaction buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt and 100 um mgcl2 ) .
a 50 l reaction was initiated and 5 l was removed and quenched at 0 , 0.5 , 1 , 2 , 4 , 8 and 16 minute time points in formamide loading buffer ( 0.025% sds , 95% formamide , 0.025% bromophenol blue , 0.025% xylene cyanol and 18 mm edta ) .
quenched samples were heated to 95c for 2 minutes and resolved by denaturing urea page .
the amount of substrate degraded ( fmol ) was plotted vs. time and linear regression was used to determine the initial rate ( slope ) of the linear portion of the curve with graphpad prism 6 .
the initial rate was converted specific activity ( nt / min / molecule of enzyme ) by dividing the initial rate ( fmol substrate processed / min ) by the fmol of enzyme used in each assay and multiplying by n-3 ( where n is the number of nucleotides of the substrate and 3 is the length of the end - product ) .
briefly , the rna coding sequence was flanked by a 5 ' hammerhead ribozyme and 3 ' hepatitis delta virus ribozyme ( hdv ) to ensure homogeneous ends .
constructs were cloned into a prsf vector containing a t7 promoter and the rna produced by run - off transcription .
the pre - let-7 sequence used in this study is pre - let7a1 : ugagguaguagguuguauaguuuuagggucacacccaccacugggagauaacuauacaaucuacugucuuuc transcribed rna was gel purified with denaturing page and resuspended in depc - treated water .
a range of dis3l2 concentrations ; 05 nm for oligou , pre - let-7-u15 , deoxyu15 and pre - let-7 substrates and 0150 nm for a15 and c15 were incubated with 1 pm 5 ' radiolabeled rna for 1 hour in binding buffer ( 20 mm hepes ph 7.5 , 50 mm nacl , 1 mm dtt , 50 g / ml bsa and 100 m edta ) .
we determined that wild - type dis3l2 is inactive in the presence of edta and no magnesium , but binds to rna with the same affinity as the inactive d389n mutant .
a slot blot filtration system was used ( biorad ) to capture dis3l2/rna complexes on the top nitrocellulose membrane and unbound rna on the bottom nylon membrane .
the membranes were washed with 100 l of binding buffer , prior to applying 100 l of the binding reaction , followed by 100 l of binding buffer . the nitrocellulose and nylon membranes were dried and analyzed by phosphor imaging .
all binding assays were conducted in triplicate , were quantified and fraction bound plotted vs. free protein concentration .
a , dis3l2 exonuclease assays were conducted with 5 ' radio - labeled rna substrates at 30c over a 16 minute time course and products resolved by denaturing urea page .
two representative gels , one from an exonuclease assay with u15 that was used to quantify dis3l2 enzymatic activity and another for deoxyu15 , showing no detectable activity under the initial rate conditions tested .
b , the initial rate plots for each substrate used to calculate specific activity as shown in fig .
the concentration of enzyme used to measure the initial rate is indicated on each plot .
the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .
dis3l2-rna complexes were captured on the top nitrocellulose membrane and unbound rna to the bottom nylon membrane .
1b , was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .
extended data figure 2 | sequence alignment of mouse and human dis3l2 , yeast rrp44 and human dis3 .
dis3l2 is composed of two cold shock domains ( csd1 , residues 49240 , 1 , 15 and csd2 , residues 241324 , 610 , 2 ) at its n - terminus followed by a catalytic rnb domain ( residues 325765 , 317 , 1121 ) and flanked by an s1 domain ( residues 766856 , 2229 ) .
the secondary structure elements deduced from the structure of mouse dis3l2 are shown on top of the sequences , color - coded by domain as in fig .
the segments of mouse dis3l2 that were truncated to facilitate crystallization are outlined in a black box .
blue diamonds indicate residues interacting with the backbone of u14 rna and red diamonds denote conserved active site residues .
extended data figure 3 | substrate specificity of truncated dis3l2 and electron density map for u14 rna . a , comparison of specific activity ( nt min molecule ) of truncated dis3l2 construct resembling the one used in crystallization , but without the d389n mutation with the same set of substrates analyzed for wild - type dis3l2 .
truncated dis3l2 had comparable levels of activity and displayed the same substrate preference as wild - type dis3l2 .
the specific activity was calculated from the initial rate plots , in the same way as described in fig.1a and extended data fig .
initial molecular replacement phases were improved by density modification with resolve and automated model building with buccaneer , as implemented by the autobuild wizard in phenix .
the unbiased density modified electron density map prior to inclusion of rna is shown contoured at 1. despite the crystallization of dis3l2 in complex with u13 , unambiguous density allowed modeling of u14 ( details are discussed in the methods ) .
clear electron density accounted for all 14 nucleotides , except some disorder contributed to weak electron density surrounding u5 , precluding its accurate placement in density .
extended data figure 4 | dis3l2 domain interface analysis . a , analysis of the csd1-rnb interface .
the conformation of csd1 is stabilized by two protein - protein interactions with the rnb ( k240 with d739 and d91 with t613 ) , and an rna mediated interaction with the rnb through u6-u7 and 11 .
csd2 is intimately associated with csd1 , but also interfaces with the rnb through 3 ( s242 with e337 and k319 with e332 ) .
s1 is part of a large hydrophobic interface with rnb ( 11 , 13 , 17 and 18 , 19 ) .
the s1 domain also interfaces with the rnb through interactions with u8 ( also see fig .
3d ) d , csd1 is further stabilized through an rna mediated interaction with s1 .
a , specific activity ( nt min molecule ) of dis3l2 with increasing length oligou ( u9 , u11 , u13 and u15 ) .
b , the initial rate plots for each substrate used to calculate specific activity in panel a. the amount of substrate degraded ( fmol ) was plotted vs. time and the initial rate ( slope ) was determined with linear regression using graphpad prism 6 .
c , equilibrium dissociation constants ( kd ) for increasing oligou length . the kd  s.d . determined from
d , the equilibrium dissociation constant ( kd ) for each substrate in panel c was determined by plotting the fraction rna bound vs. the concentration of free dis3l2 and fit by non - linear regression with graph pad prism 6 .
extended data figure 6 | dis3l2 has a conserved active site and an rna path that resembles rnase ii .
conserved active site residues are shown as green sticks and u13 and u14 are shown as orange sticks . a single mg ion ( purple sphere )
as proposed for rnase ii , dis3l2 may utilize a two mg ion mechanism during catalysis .
dis3l2 side chains and the mg ion are colored the same as in panel a , and yrrp44 residues are shown as grey sticks .
a wide - open funnel created by the csd lobe and s1 allows rna to access the  top  of the rnb .
the path of rna in rnase ii more closely resembles that in dis3l2 , compared to yrrp44 , though narrow along its length , underscoring its ability to accommodate only single - stranded rna substrates .
e , perpendicular  side  and  top  views of the electrostatic surface potential of dis3l2 ( contoured at 5 kt / e , white = neutral , blue = positive and red = negative ) .
a much wider positively charged funnel on the  top  of the rnb supports the ability of dis3l2 to degrade structured rna substrates .
f , electrostatic surface potential of the single - strand specific rnase ii , in the same configuration as panel c. a narrow rna binding channel can only accommodate single - stranded rna .
extended data figure 7 | dis3l2 discriminates u over a and c. a , dis3l2 selects oligou - tailed substrates by way of an extensive u - specific interaction network along most of its binding path . in u - zone 1
hydrogen bond pairs are shown as dashed lines and disrupted hydrogen bonds are shown as curved red lines .
b , u - zone 2 , with the same layout as panel a. c , u - zone 3 . extended data figure 8 | initial rate plots of dis3l2 u - zone mutants . a , initial rate plots for
assays were conducted under the same conditions as described for u15 in extended data fig . 1 . , where reactions contained 0.5 nm enzyme and 100 nm 5 ' radio - labeled u15 .
b , initial rate plots for u - zone 2 , conducted as described for panel a. c , initial rate plots for u - zone 3 , conducted as described for panel a. extended data figure 9 | binding affinity of dis3l2 u - zone mutants a , slot blot filter - binding assay was used to measure the binding affinity of selected dis3l2 mutants with u15 . the equilibrium dissociation constant ( kd ) for u - zone 1 mutants as shown in fig .
3 g , was determined by plotting the fraction rna bound vs. the concentration of free protein and fit by non - linear regression with graph pad prism 6 .
b , the equilibrium dissociation constant ( kd ) for u - zone 2 mutants as shown in fig .
3 g , and measured as described for panel a. c , the equilibrium dissociation constant ( kd ) for u - zone 3 as shown in fig .
 summary paragraphthe pluripotency factor lin28 inhibits the biogenesis of the let-7 family of mammalian micrornas14 . 
 lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5 , glucose metabolism6 and tissue regeneration7 . 
 alternatively , lin28 overexpression is correlated with the onset of numerous cancers8 , while let-7 , a tumor suppressor , silences several human oncogenes5 . 
 lin28 binds to precursor let-7 ( pre - let-7 ) hairpins9 , triggering the 3 ' oligo - uridylation activity of tut4/71012 . 
 the oligou tail added to pre - let-7 serves as a decay signal , as it is rapidly degraded by dis3l213,14 , a homolog of the catalytic subunit of the rna exosome . 
 the molecular basis of lin28 mediated recruitment of tut4/7 to pre - let-7 and its subsequent degradation by dis3l2 is largely unknown . to examine the mechanism of dis3l2 substrate recognition we determined the structure of mouse dis3l2 in complex with an oligou rna to mimic the uridylated tail of pre - let-7 . 
 three rna binding domains form an open funnel on one face of the catalytic domain that allows rna to navigate a path to the active site different from its exosome counterpart . 
 the resulting path reveals an extensive network of uracil - specific interactions spanning the first twelve nucleotides of an oligou - tailed rna . 
 we identify three u - specificity zones that explain how dis3l2 recognizes , binds and processes uridylated pre - let-7 in the final step of the lin28/let-7 pathway . 