Regulation of translation and mRNA stability in early mammalian development

Author(s): Freimer, Jacob W | Advisor(s): Blelloch, Robert | Abstract: Post-transcriptional regulation is critical to ensure precise control of mRNA and protein levels. Here we characterize the regulation of mRNA stability and translation, focusing on the impact of microRNAs, in two systems in early mouse development. Mouse oocyte maturation, fertilization, and reprogramming occur in the absence of transcription and thus changes in mRNA levels and translation rate are regulated through post-transcriptional mechanisms. Surprisingly, microRNA function is absent during this critical period of mammalian development. In Chapter 2 we investigated the mechanisms underlying the global suppression of microRNA activity. In both mouse and frogs, microRNA function was active in growing oocytes, but then absent during oocyte maturation. RNA-Seq of mouse oocytes uncovered that the microRNA effector protein AGO2 is predominantly expressed as an alternative isoform that encodes a truncated protein lacking all of the known essential domains. Full length Ago2 as well as the related Argonautes (Ago1, Ago3, and Ago4) were lowly expressed in maturing mouse oocytes. Reintroduction of full-length AGO2 together with an exogenous microRNA in either mouse or frog oocytes restored translational repression of a target reporter. However, levels of endogenous transcripts remained unchanged. Consistent with a lack of microRNA activity, analysis of transcripts with alternative polyadenylation sites showed increased stability of transcripts with a longer 3’ UTR during oocyte maturation. Redundant mechanisms protecting endogenous transcripts and the conserved loss of microRNA activity suggest a strong selection for suppressing microRNA function in vertebrate oocytes.Translation and mRNA degradation are intimately connected, yet the mechanisms that regulate both are not fully understood. In Chapter 3, we examine the regulation of translation and mRNA stability in mouse embryonic stem cells (ESCs) and during differentiation. In contrast to previous reports, we found that transcriptional changes account for most of the molecular changes during ESC differentiation. However, within ESCs there was a positive correlation between translation level and mRNA stability. We asked if the RNA-binding protein DDX6 links these processes in ESCs since the yeast homolog of DDX6 connects codon optimality and mRNA stability. However, there was minimal connection between codon usage and stability changes in DDX6 KO ESCs. DDX6 has also been implicated in microRNA mediated repression, a process involving both translational repression and mRNA destabilization. Surprisingly, the loss of DDX6 leads to the translational derepression of microRNA targets without affecting mRNA stability. Furthermore, DDX6 KO ESCs share overlapping phenotypes and global molecular changes with ESCs that completely lack all microRNAs. Together our results demonstrate that it is possible to decouple the two forms of microRNA induced repression and emphasize that the translational aspect of microRNA repression is underappreciated. Together these studies provide new insights into how microRNA activity is regulated as well as the downstream effectors that carry out microRNA induced translational repression.