The present communication reports, to our knowledge, the first studies of Zcchc11 in vivo
. We find that Zcchc11 is widely expressed across multiple tissues shortly after birth and that in neonatal mice Zcchc11 deficiency results in a failure to thrive, associated with diminished IGF-1 expression. Mice with a complete IGF-1 deficiency have perinatal lethality and decreased growth rates 
consistent with, but more severe than, the phenotypes observed in the Zcchc11-deficient mice. Like the Zcchc11-deficient mice, genetic engineering that reduces but does not eliminate IGF-1 signaling causes proportionally decreased growth 
. Zcchc11 deficiency results in decreased uridylation of miRNAs in the liver, including miRNAs that target the IGF-1 3′-UTR. Gene expression tied to this 3′-UTR is enhanced by the uridylation of miRNAs or the increased expression of Zcchc11. Altogether, we interpret these results as supporting a model in which the uridylation of miRNAs by Zcchc11 in the neonatal liver is essential for optimal IGF-1 expression and its promotion of growth and survival through the early postnatal period. However, Zcchc11 is a large and multifunctional protein, and we recognize that the abilities of Zcchc11 to uridylate other substrates 
and to exert uridylation-independent activities 
may additionally contribute to the complex phenotypes of Zcchc11-deficient mice.
The decreased growth rate of Zcchc11-deficient mice is complementary to the increased growth rate observed in Lin28a-overexpressing transgenic mice 
. Knockdown of Lin28 and Zcchc11 in cell lines results in similar phenotypes, and these proteins physically interact 
. These similarities support the concept that Zcchc11 and Lin28 are involved in overlapping pathways in vivo
. However, the suggestion that Zcchc11-Lin28 interactions may be essential for embryonic stem cell maintenance 
is not supported by our observation that Zcchc11 deficiency does not impact growth or survival during embryogenesis. Like Zcchc11, Lin28 proteins are particularly expressed in the tissues of young mice 
. It will be of great interest to learn whether mice with deficiencies in Lin28a, Lin28b or both have phenotypes involving perinatal lethality and decreased growth, as observed with the Zcchc11-deficient mice.
Untemplated uridines and adenines on mature miRNAs are consistent findings in deep sequencing analyses 
, but the mechanisms and significance of such terminal additions have been difficult to discern. Previous in vitro
studies had suggested that Zcchc11 is one of several enzymes capable of end-modifying mature miRNAs and that miRNA sequence variety might regulate transcript expression 
. The only other mouse model of PAP mutation, mice deficient in PAPD4/GLD-2, has no reported growth or survival phenotype 
. Along with our data that other PAPs were expressed in the neonatal livers of the Zcchc11-deficient mice, these findings conclusively demonstrate that the different PAPs have unique and non-overlapping roles in vivo
. Unlike GLD-2, Zcchc11 is critical for thriving through the neonatal period, likely due in part to its ability to enhance hepatic IGF-1 expression.
In embryonic stem cell lines, Zcchc11 knockdown increases let-7 levels, due to Zcchc11-mediated uridylation of precursors 
. The Zcchc11-deficient mice allowed the examination of miRNA regulation in primary cells of living animals, and they revealed that Zcchc11 is not an essential determinant of mature let-7 or any mature miRNA quantity in the neonatal liver. Furthermore, insertional mutagenesis of Zcchc11 did not increase let-7 quantities in primary embryonic stem cells derived from these mice. The relationships among these terminal uridyltransferases (Zcchc11 and Zcchc6), Lin28 proteins (a and b), and let-7 miRNAs in embryonic stem cells are complex and dynamic 
. The present data show that Zcchc11 is not an absolute requirement for stem cell maintenance or low levels of let-7. There may be other conditions in which precursor uridylation by Zcchc11 is essential to regulating let-7, such as perhaps early embryogenesis when Lin28a is especially active. This mouse model will serve as a useful resource for determining if and when this uridyltransferase enzyme may influence miRNA biogenesis or content.
In contrast to the unchanged abundance of miRNAs in the liver, mature miRNA lengths and sequences were altered by Zcchc11 deficiency. Mature miRNAs in the liver were longer and more likely to end in uridine, including untemplated uridines, when Zcchc11 was present. Thus, our data show that Zcchc11 functions to uridylate mature miRNAs in vivo.
In addition to providing unprecedented evidence of an enzyme actively uridylating the 3′ terminus of miRNAs in vivo
, these mice yield new insights into the scope of Zcchc11 modification of the miRNome. Rather than targeting only one individual miRNA, as has been previously documented for Zcchc11 and other PAPs 
, we show here that Zcchc11 targets the 3′ terminus of multiple miRNAs. This broad substrate repertoire dramatically increases the potential targeting power of Zcchc11. Importantly, most of the end-modifications observed varied by a small number of terminal nucleotides, and we observed that even a single uridine addition was sufficient to alleviate silencing activity. Such mono-uridylation by Zcchc11 appears to distinguish the effect of Zcchc11 on mature miRNAs from that described for pre-miRNAs, which is processive and results in a string of uridines being added 
. Our data further expand our understanding of the molecular implications of miRNA uridylation by demonstrating that coordination of miRNA uridylation events across a 3′-UTR have combinatorial effects. The ability to adjust the silencing activity of many miRNAs targeting one transcript provides a wide dynamic range for enhancing gene expression. The exonuclease Nibbler was recently identified as capable of shortening miRNAs by removing terminal nucleotides 
. Such enzymes may counter-balance the nucleotidyltransferase activities of PAPs like Zcchc11. The abundance and remarkable stability of miRNAs suggest that mechanisms regulating miRNA activity are crucial, but they are only beginning to be elucidated 
We propose that the miRNA 3′ terminus functions as a critical regulatory node that is remodeled by diverse enzymes to adjust miRNA silencing and tune gene expression. The present results support this nascent paradigm by demonstrating essential in vivo roles of Zcchc11 in miRNome remodeling and postnatal development. Zcchc11 mediates mature miRNA uridylation, facilitates hepatic IGF-1 expression, and enhances growth and survival through the neonatal period.