Functional roles for the terminal uridyltransferase enzymes Zcchc6 and Zcchc11 in mammalian biology

Abstract

Diverse species of RNA can be post-transcriptionally uridylated on 3’ termini to modulate their biological function. Key cellular enzymes involved in the catalysis of RNA uridylation have been identified as two closely related terminal uridyltransferase (TUT) family members known as Zcchc6 and Zcchc11. To date, most reports on the function of TUTs have been gleaned from reductionist in vitro systems, however the biological function of these enzymes in integrated animal models remains unknown. The goal of this work was to investigate physiological roles for the TUT proteins during normal homeostasis and during infectious stress. To achieve this, we generated two constitutive, animal models of TUT-deficiency for both Zcchc6 and Zcchc11. The first knockout animal of the two TUTs to be examined was Zcchc11. Given previous reports that Zcchc11 played essential roles in stem cell biology, it was anticipated that Zcchc11-null mice would be result in embryonic lethality. We observed decreased survival and organismal growth following birth. The hepatic small RNA transcriptome revealed reduced sequence lengths and terminal uridylation across mature microRNAs and the expression of IGF-1 was enhanced by Zcchc11 expression in vitro. MiRNA silencing of IGF-1 was alleviated by the uridylation of IGF-targeting miRNA. We concluded that the Zcchc11-mediated terminal uridylation of mature microRNAs is pervasive and physiologically significant. In the second model examined, we observed that Zcchc6 deficiency did not impact perinatal mortality or litter size, but in contrast to Zcchc11-null mice, Zcchc6-null animals exhibited enhanced organismal growth following birth. A survey of tissue Zcchc6 mRNA expression showed enrichment in the lungs and upon further analysis we observed Zcchc6 to be uniquely and highly expressed in mouse and human primary alveolar and bone marrow derived macrophages, increased during monocyte-to-macrophage differentiation and regulated the expression of select cytokines including IL-6 and CXCL1 during inflammation. These studies indicated that Zcchc6 was required for the development of immunocompetent macrophages and calibrated macrophage-mediated innate immune responses in the airspaces. Taken together, these data provide evidence of independent and overlapping roles for TUT proteins in diverse physiological systems in living animal models.