Novel Roles for the Terminal RNA Uridylyltransferases ZCCHC6 and ZCCHC11 in Development and Disease

Abstract

Terminal RNA uridylyltransferases (TUTases) are a class of non-canonical polyadenylate polymerases that are thought to function primarily by adding non-templated uridines to the 3’ ends of a wide range of RNA targets. Recently, two TUTases, ZCCHC6 and ZCCHC11, were shown to uridylate mRNAs, thereby marking them for degradation. While RNA uridylation is emerging as a potent mechanism of post-transcriptional gene regulation, uridylation-independent functions of TUTases have also been reported. Despite these insights, the biological importance of ZCCHC6/11 in physiologic and pathologic settings remains poorly understood. This dissertation explores the role of these TUTases in normal development and in cancer. First, we examine Zcchc6/11 function in development and differentiation by profiling TUTase expression during mouse embryogenesis. In many tissues, we observe robust TUTase expression that decreases across developmental time, indicating a potential regulatory role. Using in vitro models of murine myogenesis, we find that Zcchc6/11 are regulators of muscle differentiation. Surprisingly, mutational analysis of Zcchc6 reveals that its catalytic activity is dispensable, while a portion of its N-terminal domain is required for this effect, indicating uridylation-independent mechanisms. Similarly, in mouse embryonic stem cells, TUTase downregulation contributes to the exit from naïve pluripotency, suggesting a broader regulatory role in cellular differentiation. Collectively, these data indicate that TUTases regulate differentiation across diverse tissues and developmental stages. Next, we investigate the impact of ZCCHC6/11 in cancer. Expression profiling reveals low levels of the TUTases in most normal adult tissues, yet robust expression across a variety of cancer cell lines and primary tumors. Consistent with an oncofetal pattern of expression, many adult tissues where TUTase expression is absent also exhibit reactivation to fetal levels following malignant transformation. Functionally, we demonstrate that ZCCHC6 supports the growth and tumorigenicity of a subset of cancer cell lines. Mechanistically, these effects are associated with altered mRNA turnover, including dysregulation of cell cycle factors and histone proteins. Finally, we find that CRISPR-Cas9 mediated knockout of ZCCHC6 sensitizes cancer cells to DNA damaging agents, potentially through the disruption of histone turnover. Overall, our results uncover novel functional roles for ZCCHC6/11, with therapeutic implications for both tissue regeneration and cancer.