to gain insight into how lin28 controls let-7 maturation , we analyzed by mass spectrometry affinity purified lin28-containing protein complexes from mouse es cells that were engineered to contain a doxycycline - inducible flag - lin28 transgene . we performed flag - immunoprecipitations from control ( dox ) and induced ( + dox , 48hrs ) cell extracts . we subjected the affinity eluate to sds - polyacrylamide gel electrophoresis followed by flag western blot ( supplementary fig . zcchc11 is predicted to contain two poly(a)polymerase domains , a nucleotidyltransferase ( ntr ) domain and several cchc - type zinc finger domains ( fig . this non - canonical poly(a)polymerase is present in seven mouse proteins of which some have been shown to catalyze terminal uridine instead of adenosine additions . these proteins include tut1 that is essential for u6 snrna recycling26 . additionally , two tutasae , papd1 and papd5 , terminally uridylate histone h1 mrna , leading to its turnover27 . as such , research on terminal uridylation is still in its infancy and our knowledge about the functional consequences of this posttranscriptional modification is limited . to examine whether zcchc11 can directly catalyze the terminal oligonucleotide addition to pre - let-7 and to assess the potential functional interaction between lin28 and zcchc11 , we developed an in vitro uridylation assay . first , pre - let-7 rna binding was monitored by electromobility shift assays ( emsas ) to confirm that both the affinity - purified flag - lin28 from es cells and recombinant his6-lin28 , were functional in pre - let-7 binding and to ensure that sufficient amounts of lin28 from these two sources were used in subsequent uridylation assays ( fig . for in vitro uridylation assays , we incubated synthetic pre - let-7 rna with the affinity - purified proteins in the presence of [ -p]-utp . e. coli poly(a ) polymerase served as a positive control in these reactions , since this enzyme can also catalyze 3 terminal uridine addition in the absence of atp . though we observed the weak appearance of some uridylated pre - let-7a when ha - zcchc11 alone was added to the reaction , this activity was greatly enhanced by addition of either flag - lin28 purified from es cells or recombinant lin28 , thus indicating that lin28 directly recruits the zcchc11 tutase activity to pre - let-7a ( fig . neither flag - lin28 nor recombinant lin28 alone possessed any detectable uridylation activity , confirming that the appearance of the uridylated product was dependent on the presence of zcchc11 in the reaction . we also resolved products of the in vitro uridylation assays on high resolution sequencing gels . this analysis indicated that purified ha - zcchc11 when incubated with recombinant lin28 primarily catalyzed the addition of 4 - 8 uridines to the synthetic pre - let-7a-1 rna ( supplementary fig . these competition assays performed with synthetic pre - let-7 g rna revealed that unlabeled utp is able to compete effectively in these reactions and leads to a diminution of detectable tutase activity whereas the presence of atp , gtp , and ctp did not . these results demonstrate that the zcchc11 preferentially utilizes utp for its catalytic activity ( fig . next , we generated a mutant version of the zcchc11 enzyme in which two aspartate residues ( d1008 , d1010 ) from the catalytic triad in the ntr domain were mutated to alanines . unlike wild - type zcchc11 , the mutant protein showed no uridylation activity towards pre - let-7a , showing that the pre - let-7 uridylation requires a catalytically active zcchc11 enzyme in these reactions ( fig . , reactions were performed using a synthetic pre - mir-21 as a substrate . as with pre - let-7a and pre - let-7 g , we observed a minimal background uridylase activity in reactions containing zcchc11 alone ; however , unlike reactions performed with pre - let-7 , this activity was not enhanced by the addition of lin28 . in control reactions these experiments illustrate that lin28-mediated tutase activity is selective for pre - let-7 family members . next , we performed knockdown experiments in mouse embryonic cells and monitored the effect of zcchc11 depletion on the levels of mature mirnas by real - time pcr . we transfected p19 embryonal carcinoma ( ec ) cells with at least two sirnas for lin28 , zcchc11 , or zcchc6 ( a closely related family member ) . upon either lin28 or zcchc11 knockdown , we found that the levels of mature let-7a and let-7 g increased while the control mirna , mir-21 , was unaffected ( fig . importantly , we verified that knockdown of zcchc11 does not affect lin28 levels , thereby excluding the possibility that the observed effect on let-7 levels is due to zcchc11 regulation of lin28 expression ( fig . 3a ) . in multiple independent experiments ( fig . 3 , 4 , supplementary fig . 3 , and data not shown ) we observed a greater increase in let-7 levels upon lin28 depletion compared to that of zcchc11 knockdowns . this observation could be explained by the more efficient knockdown that was achieved for lin28 than for zcchc11 . however , this observation is also consistent with the notion that lin28 can block let-7 processing at multiple steps of the let-7 processing pathway , some of which may be independent of zcchc11 function . for example , lin28 has been reported to inhibit the microprocessor leading to a block at the pri - let-7 to pre - let-7 processing step6 - 9 . we extended our studies using a panel of sirnas to target lin28 , lin28b , and five of the seven known or predicted tutases ( zcchc11 , zcchc6 , pols , papd4 , and papd5)24,25 . we excluded tut1 , the u6 snrna - specific tutase , from our analysis due to the reported cell lethality caused by its depletion by rnai26 . this analysis confirmed that only reduction in lin28 or zcchc11 reproducibly and substantially elevated let-7a levels ( supplementary fig . 3 ) . this result highlights that zcchc11 is the key tutase responsible for regulating levels of let-7a in p19 cells . we explored the relevance of this regulatory pathway in mouse es cells using sirnas to deplete zcchc11 . we found that zcchc11 depletion led to an increase in mature let-7a and let-7 g levels , but had no effect on levels of mir-21 ( fig . moreover , overexpression of the catalytically inactive mutant zcchc11 where two of three aspartates of the catalytic triad are mutated to alanine also resulted in increased let-7 levels ( fig . these results showed that zcchc11 mediates the selective inhibition of let-7 maturation in es cells . to investigate more broadly the impact of zcchc11 on mirna regulation , global profiling of mature mirna expression we found that multiple let-7 family members ( let-7b , let-7d , let-7 g , and let-7i ) were highly and reproducibly elevated by knockdown of both lin28 and zcchc11 . this global analysis demonstrates unequivocally that zcchc11 is dependent in large measure for lin28-mediated repression . to address further the in vivo significance of this regulatory pathway , we used reporter assays to monitor the repression of let-7 responsive target genes . in these experiments , we co - transfected p19 embryonal carcinoma cells with renilla luciferase constructs containing either fully complementary , partially complementary , or mutant let-7 binding sites in the 3utr , together with a firefly luciferase control plasmid . sirna - mediated depletion of either lin28 or zcchc11 in these cells led to the specific repression of reporter constructs containing let-7 target sites . hence , the specific accumulation of mature let-7 mirna we observed by either lin28 or zcchc11 depletion ( fig . 3 and 4a ) leads to the effective repression of reporter constructs containing let-7 target sites . we found that the extent of reporter gene repression was comparable between lin28-depleted and zcchc11-depleted cells . furthermore , overexpression of the inactive mutant zcchc11 protein also resulted in repression of the let-7 responsive reporter constructs , whereas overexpression of wild - type zcchc11 did not ( fig . these data demonstrate that the presence of catalytically active zcchc11 is essential for the posttranscriptional control of let-7 maturation and may provide embryonic cells with a mechanism that enables expression of let-7 target genes . to gain insight into how lin28 controls let-7 maturation , we analyzed by mass spectrometry affinity purified lin28-containing protein complexes from mouse es cells that were engineered to contain a doxycycline - inducible flag - lin28 transgene . we performed flag - immunoprecipitations from control ( dox ) and induced ( + dox , 48hrs ) cell extracts . we subjected the affinity eluate to sds - polyacrylamide gel electrophoresis followed by flag western blot ( supplementary fig . zcchc11 is predicted to contain two poly(a)polymerase domains , a nucleotidyltransferase ( ntr ) domain and several cchc - type zinc finger domains ( fig . this non - canonical poly(a)polymerase is present in seven mouse proteins of which some have been shown to catalyze terminal uridine instead of adenosine additions . these proteins include tut1 that is essential for u6 snrna recycling26 . additionally , two tutasae , papd1 and papd5 , terminally uridylate histone h1 mrna , leading to its turnover27 . as such , research on terminal uridylation is still in its infancy and our knowledge about the functional consequences of this posttranscriptional modification is limited . to examine whether zcchc11 can directly catalyze the terminal oligonucleotide addition to pre - let-7 and to assess the potential functional interaction between lin28 and zcchc11 , we developed an in vitro uridylation assay . first , pre - let-7 rna binding was monitored by electromobility shift assays ( emsas ) to confirm that both the affinity - purified flag - lin28 from es cells and recombinant his6-lin28 , were functional in pre - let-7 binding and to ensure that sufficient amounts of lin28 from these two sources were used in subsequent uridylation assays ( fig . for in vitro uridylation assays , we incubated synthetic pre - let-7 rna with the affinity - purified proteins in the presence of [ -p]-utp . e. coli poly(a ) polymerase served as a positive control in these reactions , since this enzyme can also catalyze 3 terminal uridine addition in the absence of atp . though we observed the weak appearance of some uridylated pre - let-7a when ha - zcchc11 alone was added to the reaction , this activity was greatly enhanced by addition of either flag - lin28 purified from es cells or recombinant lin28 , thus indicating that lin28 directly recruits the zcchc11 tutase activity to pre - let-7a ( fig . neither flag - lin28 nor recombinant lin28 alone possessed any detectable uridylation activity , confirming that the appearance of the uridylated product was dependent on the presence of zcchc11 in the reaction . we also resolved products of the in vitro uridylation assays on high resolution sequencing gels . this analysis indicated that purified ha - zcchc11 when incubated with recombinant lin28 primarily catalyzed the addition of 4 - 8 uridines to the synthetic pre - let-7a-1 rna ( supplementary fig . to elucidate the ribonucleotide selectivity of zcchc11 , we performed uridylation assays in the presence of excess non - radiolabeled nucleotides . these competition assays performed with synthetic pre - let-7 g rna revealed that unlabeled utp is able to compete effectively in these reactions and leads to a diminution of detectable tutase activity whereas the presence of atp , gtp , and ctp did not . these results demonstrate that the zcchc11 preferentially utilizes utp for its catalytic activity ( fig . next , we generated a mutant version of the zcchc11 enzyme in which two aspartate residues ( d1008 , d1010 ) from the catalytic triad in the ntr domain were mutated to alanines . unlike wild - type zcchc11 , the mutant protein showed no uridylation activity towards pre - let-7a , showing that the pre - let-7 uridylation requires a catalytically active zcchc11 enzyme in these reactions ( fig . , reactions were performed using a synthetic pre - mir-21 as a substrate . as with pre - let-7a and pre - let-7 g , we observed a minimal background uridylase activity in reactions containing zcchc11 alone ; however , unlike reactions performed with pre - let-7 , this activity was not enhanced by the addition of lin28 . in control reactions , these experiments illustrate that lin28-mediated tutase activity is selective for pre - let-7 family members . next , we performed knockdown experiments in mouse embryonic cells and monitored the effect of zcchc11 depletion on the levels of mature mirnas by real - time pcr . we transfected p19 embryonal carcinoma ( ec ) cells with at least two sirnas for lin28 , zcchc11 , or zcchc6 ( a closely related family member ) . upon either lin28 or zcchc11 knockdown , we found that the levels of mature let-7a and let-7 g increased while the control mirna , mir-21 , was unaffected ( fig . importantly , we verified that knockdown of zcchc11 does not affect lin28 levels , thereby excluding the possibility that the observed effect on let-7 levels is due to zcchc11 regulation of lin28 expression ( fig . 3a ) . in multiple independent experiments ( fig . 3 , 4 , supplementary fig . 3 , and data not shown ) we observed a greater increase in let-7 levels upon lin28 depletion compared to that of zcchc11 knockdowns . this observation could be explained by the more efficient knockdown that was achieved for lin28 than for zcchc11 . however , this observation is also consistent with the notion that lin28 can block let-7 processing at multiple steps of the let-7 processing pathway , some of which may be independent of zcchc11 function . for example , lin28 has been reported to inhibit the microprocessor leading to a block at the pri - let-7 to pre - let-7 processing step6 - 9 . we extended our studies using a panel of sirnas to target lin28 , lin28b , and five of the seven known or predicted tutases ( zcchc11 , zcchc6 , pols , papd4 , and papd5)24,25 . we excluded tut1 , the u6 snrna - specific tutase , from our analysis due to the reported cell lethality caused by its depletion by rnai26 . this analysis confirmed that only reduction in lin28 or zcchc11 reproducibly and substantially elevated let-7a levels ( supplementary fig . this result highlights that zcchc11 is the key tutase responsible for regulating levels of let-7a in p19 cells . we explored the relevance of this regulatory pathway in mouse es cells using sirnas to deplete zcchc11 . we found that zcchc11 depletion led to an increase in mature let-7a and let-7 g levels , but had no effect on levels of mir-21 ( fig . , overexpression of the catalytically inactive mutant zcchc11 where two of three aspartates of the catalytic triad are mutated to alanine also resulted in increased let-7 levels ( fig . these results showed that zcchc11 mediates the selective inhibition of let-7 maturation in es cells . to investigate more broadly the impact of zcchc11 on mirna regulation , global profiling of mature mirna expression was used to analyze rna isolated from both lin28 and zcchc11 knockdowns ( fig . we found that multiple let-7 family members ( let-7b , let-7d , let-7 g , and let-7i ) were highly and reproducibly elevated by knockdown of both lin28 and zcchc11 . this global analysis demonstrates unequivocally that zcchc11 is dependent in large measure for lin28-mediated repression . to address further the in vivo significance of this regulatory pathway , we used reporter assays to monitor the repression of let-7 responsive target genes . in these experiments , we co - transfected p19 embryonal carcinoma cells with renilla luciferase constructs containing either fully complementary , partially complementary , or mutant let-7 binding sites in the 3utr , together with a firefly luciferase control plasmid . sirna - mediated depletion of either lin28 or zcchc11 in these cells led to the specific repression of reporter constructs containing let-7 target sites . hence , the specific accumulation of mature let-7 mirna we observed by either lin28 or zcchc11 depletion ( fig . 3 and 4a ) leads to the effective repression of reporter constructs containing let-7 target sites . we found that the extent of reporter gene repression was comparable between lin28-depleted and zcchc11-depleted cells . furthermore , overexpression of the inactive mutant zcchc11 protein also resulted in repression of the let-7 responsive reporter constructs , whereas overexpression of wild - type zcchc11 did not ( fig . these data demonstrate that the presence of catalytically active zcchc11 is essential for the posttranscriptional control of let-7 maturation and may provide embryonic cells with a mechanism that enables expression of let-7 target genes . here , we describe the first example of a specific terminal uridylyl transferase ( tutase ) that controls mature mirna levels . we demonstrate that zcchc11 is the enzyme responsible for lin28-mediated pre - let-7 uridylation and blockade of let-7 processing in es cells . we have shown that the pre - let-7 tutase activity of zcchc11 is lin28 dependent and that zcchc11 exclusively regulates the expression of the let-7 mirna family . work from miska and colleagues has identified a homologous pathway in c. elegans indicating that this novel mirna gene regulatory mechanism is evolutionarily conserved ( ref ) . based on the substoichiometric and salt sensitive nature of the lin28-zcchc11 interaction , we propose a model in which lin28 binding to pre - let-7 recruits zcchc11 to add the u - tail . there is emerging evidence supporting extensive posttranscriptional control of mirna expression in various developmental and disease contexts . it will therefore be of interest to explore whether this type of regulatory mechanism involving the recruitment of a specific tutase to mirna precursors is relevant to other mirnas and to identify the enzymes responsible . previously , the only example of terminal uridylation shown to control the levels of an endogenous mammalian transcript was that of papd1 and papd5 which leads to histone mrna degradation25,27 . similarly in s. pombe , it has recently been demonstrated that cid1-mediated uridylation leads to mrna decapping and accelerated decay30 . we envision a similar downstream pathway leading to targeted decay of the uridylated pre - let-7 . consistent with an earlier report9 , we have been unable to detect robustly uridylated pre - let-7 in vivo on freshly isolated rna . although pre - let-7 with 3-terminal u - tails can inhibit dicer processing activity in vitro , we believe that based on the relative scarcity of this species in vivo , that the normal pathway likely involves the recruitment of specific nucleases leading to the rapid decay of the uridylated pre - let-7 rnas . understanding the exact downstream mechanism(s ) and identification of the nucleases involved remain important avenues for future investigation . our results provide novel insight into the role of pre - let-7 terminal uridylation as part of the lin28-mediated blockade of let-7 mirna processing in es cells and may serve as a paradigm for other mirna families . given the recent description of both lin28 and lin28b as proto - oncogenes20 - 23 and the corresponding role of the let-7 mirnas as tumor suppressors , our discovery of the tutase zcchc11 suggests a putative novel therapeutic target in cancers in which lin28 or lin28b restrict the expression of the tumor suppressor let-7 mirnas , and potentially , other diseases driven by let-7 mirna dysregulation .
lin28 and lin28b , two developmentally regulated rna - binding proteins and proto - oncogenes , selectively inhibit the maturation of let-7 family mirnas in embryonic stem ( es ) cells and certain cancers . 
 moreover , let-7 precursors ( pre - let-7 ) were previously found to be terminally uridylated in a lin28-dependent fashion . here 
 , we identify zcchc11 ( zinc finger , cchc domain containing 11 ) as the 3 terminal uridylyl transferase ( tutase ) responsible for lin28-mediated pre - let-7 uridylation and blockade of let-7 processing in mouse es cells . 
 we demonstrate that zcchc11 activity is utp - dependent , selective for let-7 , and recruited by lin28 . 
 furthermore , knockdown of either zcchc11 or lin28 , or overexpression of a catalytically inactive tutase , relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 mirnas and repression of let-7 target reporter genes . 
 our results establish a novel role for zcchc11-catalyzed pre - let-7 uridylation in the control of mirna biogenesis .