| Mammalian neurogenesis is a vital process during fetal development and persists throughout adult life. Extensive studies have revealed the roles of transcription, epigenetics, and cell-cell signaling in regulating neurogenesis, but little is known about post-transcriptional regulation which delivers a much more rapid and subcellularly localized control of gene expression. In this thesis, I studied the post-transcriptional regulation of mouse neurogenesis by two RNA binding proteins, Pumilio (Pum) 1 and 2, the two murine members of the evolutionarily conserved PUF RNA binding protein family. PUF proteins are known to mediate post-transcriptional regulation in stem cells and development in lower organisms including Drosophila, C. elegans and, the planarian Dugesia japonica, but their function in mammalian neurogenesis and their molecular mechanisms have not yet been well characterized. To investigate the roles of Pum1 and Pum2 in neurogenesis, we generated Nestin-cre-driven neural specific conditional Puml; Pum2 double knockout mice. Nestin-Cre begins to express around embryonic day 8.5 and thus results in the ablation of Pum1 and Pum2 expression in the entire central nervous system by embryonic day 15.5.;In these Puml/Pum2 double knockout mice, I observed a dramatically smaller size of the dentate gyrus (DG) in neonatal and adult brains, an indicator of the severely reduced number of neural stem cells (NSCs) after birth, associated with a drastically increased perinatal apoptosis. Consistently, in neurosphere assays, the Puml/Pum2- mutant DG give rise to fewer NSCs, that display defects in proliferation, survival and differentiation in vitro. Furthermore, using markers for different neural cell types I found that deletion of Pum1 and Pum2 led to accumulation of Tbr2-positive neuronal progenitors while fewer DCX-positive immature neurons were generated. Neurogenesis in DG generates new neurons to be incorporated into the hippocampus and this process is vital for learning and memory. Consistent with the observed defects in neurogenesis, the behavioral tests revealed that the Puml/Pum2- mutant mice were strongly impaired in learning and memory. All these lines of evidence indicate that Pum1 and Pum2 together play a major role in hippocampal neurogenesis.;To identify Pum target mRNAs and downstream pathways, I performed Pum1 and Pum2 crosslinking immunoprecipitation (iCLIP) assays in neonatal brain lysates. I was able to detect the binding sites of Pum1 and Pum2 to individual nucleotide resolution and thus revealed the preference of both Pum1 and Pum2 binding to the 3' UTR of their targets. 1,874 and 875 Pum1- and Pum2- target mRNAs were identified respectively, with 694 as common targets that are involved in multiple pathways crucial to neurogenesis, such as cell adhesion, migration, proliferation, differentiation, and apoptosis. RNA-sequencing, quantitative PCR and immunoblotting assays indicated that, in Pum1 and Pum2 single and double knockout neonatal brains, the majority of Pum targets were upregulated at the protein level, while they remained unchanged at the transcriptional level, suggesting that they were post-transcriptionally regulated by Pum. Hence, Pum1 and Pum2 achieve their neurogenic function by post-transcriptionally regulating their RNA targets in pathways that prevent both apoptosis and precocious differentiation.;Finally, I discovered that FMRP (Fragile X Mental Retardstion Protein), another important RNA binding protein in the nervous system, was a protein partner of Pum. Pum and FMRP co-migrated in protein fractionation assays and co-immunoprecipitated in an RNA- dependent manner. This indicates that Pum proteins might form collaborative networks with FMRP and possibly other post-transcriptional regulators to regulate neurogenesis.;Overall my thesis work is a contribution to our understanding of mammalian neurogenesis by uncovering the pleiotropic role of Pum1 and Pum2 post-transcriptional regulators and identifying their targeting pathways. Moreover, my finding that Pum proteins interacted with FMRP opens up the possibilities that multiple RNA binding proteins might form networks and Pum proteins could serve as "facilitators" for other molecules to bind to the RNAs. |
